Crosslinked dextran and crosslinked dextran-poly alpha-1,3-glucan graft copolymers

ABSTRACT

Compositions are disclosed herein comprising one or more crosslinked dextrans or crosslinked dextran-poly alpha-1,3-glucan graft copolymers. Further disclosed are processes for preparing such crosslinked materials, as well as their use in absorption applications.

This application claims the benefit of U.S. Provisional Application Nos.62/459,800 (filed Feb. 16, 2017) and 62/489,500 (filed Apr. 25, 2017),which are both incorporated herein by reference in their entirety.

FIELD

This disclosure is in the field of polysaccharides. For example, thisdisclosure pertains to the production of dextran-poly alpha-1,3-glucangraft copolymers that are crosslinked, and use thereof in compositionshaving advantageous aqueous liquid absorption features.

BACKGROUND

Superabsorbent materials generally are capable of absorbing high amountsof aqueous liquid, typically equivalent to many times their own weight,and retaining aqueous liquid under elevated pressure. Absorbent productsin which superabsorbent materials are commonly applied include diapers,training pants, adult incontinence products and feminine care products.The use of superabsorbent materials in these and other products increasetheir absorbent capacity while reducing their overall bulk.

Most superabsorbent materials used today are composed of crosslinkedsynthetic polymers, and are termed superabsorbent polymers (SAPs). Theseinclude, for example, polymers and co-polymers of acrylic acid oracrylamide. Despite their advantage of superabsorbency, most commercialSAPs have significant drawbacks, such as not being derivable fromrenewable sources and/or lacking sufficient biodegradability.

Certain polysaccharide compositions have been used in superabsorptionapplications, potentially taking advantage of the general renewabilityand biodegradability of these bio-derived components. For example, U.S.Pat. No. 3345358 describes gel-forming polysaccharide derivativescomprising carboxymethyl starch. As another example, highly swollen gelparticles containing a water-soluble, alkali metal salt of carboxymethylcellulose have been described in U.S. Pat. No. 2639239.

Despite these past advances, further development of polysaccharide-basedcompositions is desired for acquiring enhanced superabsorption function.

SUMMARY

In one embodiment, the disclosure concerns a composition comprising acrosslinked graft copolymer, wherein the graft copolymer portion of thecrosslinked graft copolymer comprises: (i) a backbone comprisingdextran, and (ii) poly alpha-1,3-glucan side chains comprising at leastabout 50% alpha-1,3-glucosidic linkages.

In another embodiment, the disclosure concerns a method of producing acrosslinked graft copolymer, the method comprising: (a) contacting atleast a solvent, a crosslinking agent, and a graft copolymer, whereinthe graft copolymer comprises: (i) a backbone comprising dextran, and(ii) poly alpha-1,3-glucan side chains comprising at least about 50%alpha-1,3-glucosidic linkages, whereby a crosslinked graft copolymer isproduced; and (b) optionally, isolating the crosslinked graft copolymerproduced in step (a).

In another embodiment, the disclosure concerns a composition comprisingcrosslinked dextran, wherein the dextran comprises: (i) about 87-93 wt%glucose linked at positions 1 and 6; (ii) about 0.1-1.2 wt% glucoselinked at positions 1 and 3; (iii) about 0.1-0.7 wt% glucose linked atpositions 1 and 4; (iv) about 7.7-8.6 wt% glucose linked at positions 1,3 and 6; and (v) about 0.4-1.7 wt% glucose linked at: (a) positions 1, 2and 6, or (b) positions 1, 4 and 6; wherein the weight-average molecularweight (Mw) of the dextran is about 50-200 million Daltons.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES

FIG. 1 : Example of a portion of a dextran-poly alpha-1,3-glucan graftcopolymer that can be used to prepare a crosslinked graft copolymer aspresently disclosed. In this particular illustration, a polyalpha-1,3-glucan chain (“glucan graft”) is synthesized by aglucosyltransferase enzyme (GTF) off of a pendant glucose that isalpha-1,4-linked to a dextran backbone.

FIG. 2 : Graphical representation of an example of a dextran-polyalpha-1,3-glucan graft copolymer that can be used to prepare acrosslinked graft copolymer. The dextran backbone and polyalpha-1,3-glucan side chains are presented roughly to scale with eachother. For example, the backbone can be about 1000 DPw, while each sidechain can be about 1000 DPw.

FIG. 3 . A graph illustrates the effect of starting dextranconcentration (g/L) on the DPw of dextran-poly alpha-1,3-glucan graftcopolymer produced in 2-hr and 24 hr-glucosyltransferase reactions.Refer to Example 2.

FIG. 4 shows a photograph of a dextran-poly alpha-1,3-glucan graftcopolymer sample containing 10.6 wt% dextran. Refer to Example 6.

FIG. 5 shows a photograph of a dextran-poly alpha-1,3-glucan graftcopolymer sample containing 0.9 wt% dextran. Refer to Example 6.

DETAILED DESCRIPTION

The disclosures of all cited patent and non-patent literature areincorporated herein by reference in their entirety.

Unless otherwise disclosed, the terms “a” and “an” as used herein areintended to encompass one or more (i.e., at least one) of a referencedfeature.

Where present, all ranges are inclusive and combinable, except asotherwise noted. For example, when a range of “1 to 5” is recited, therecited range should be construed as including ranges “1 to 4”, “1 to3”, “1-2”, “1-2 & 4-5”, “1-3 & 5”, and the like. Any list of possibleamounts/percentages herein can be used to describe a range, where arange can be set between any two amounts/percentages in the list.

The term “copolymer” herein refers to a polymer comprising at least twodifferent types of alpha-glucan, such as dextran and polyalpha-1,3-glucan.

The terms “graft copolymer”, “branched copolymer” and the like hereingenerally refer to a copolymer comprising a “backbone” (or “main chain”)and side chains branching from the backbone. The side chains arestructurally distinct from the backbone. Examples of graft copolymersherein are dextran-poly alpha-1,3-glucan graft copolymers, whichcomprise a backbone comprising dextran, and side chains of polyalpha-1,3-glucan. In some aspects, a dextran backbone can have a polyalpha-1,3-glucan extension, since the non-reducing end of dextran canprime poly alpha-1,3-glucan synthesis by a glucosyltransferase enzyme. Abackbone can thus be a dextran-poly alpha-1,3-glucan linear copolymer insome instances. A backbone in some aspects can itself be a branchedstructure as disclosed below; the addition of poly alpha-1,3-glucan tosuch a backbone increases the branching of the original branchedstructure.

The term “crosslinked graft copolymer” and other like terms herein referto a graft copolymer such as a crosslinked dextran-poly alpha-1,3-glucangraft copolymer. The term “crosslink” and other like terms hereintypically refer to one or more bonds (covalent and/or non-covalent) thatconnect polymers. A crosslink having multiple bonds typically comprisesone or more atoms that are part of a crosslinking agent that was used toform the crosslink. Non-covalent bonds in some aspects can be throughionic, hydrophobic, H-bonding, or van der Waals interactions.

The terms “crosslinking agent”, “crosslinker” and the like herein referto an atom or compound that can create crosslinks between glucanpolymers (e.g., poly alpha-1,3-glucan, dextran). A crosslinking agent intypical embodiments have groups that can react with hydroxyl groups ofglucose monomers of a graft copolymer.

The term “crosslinking reaction” and like terms (e.g., “crosslinkingcomposition”, “crosslinking preparation”) herein typically refer to areaction comprising at least a solvent, a crosslinking agent, and agraft copolymer. A crosslinking reaction in some aspects comprises anaqueous solvent such as water, whereas in other aspects the solvent isnon-aqueous.

The terms “poly alpha-1,3-glucan side chain” and “poly alpha-1,3-glucanbranch” (and like terms) can be used interchangeably herein. A polyalpha-1,3-glucan side chain is typically an extension of a dextranbranch (e.g., pendant glucose or short chain), since a dextran branchhas a non-reducing end that can prime poly alpha-1,3-glucan synthesis bya glucosyltransferase enzyme.

“Poly alpha-1,3-glucan homopolymer” and like terms as used herein referto poly alpha-1,3-glucan that is not part of (i) a graft copolymer or(ii) a dextran-poly alpha-1,3-glucan linear copolymer.

The terms “alpha-glucan”, “alpha-glucan polymer” and the like are usedinterchangeably herein. An alpha-glucan is a polymer comprising glucosemonomeric units linked together by alpha-glucosidic linkages. Dextranand poly alpha-1,3-glucan are examples of alpha-glucans.

The terms “glycosidic linkage”, “glycosidic bond” and the like are usedinterchangeably herein and refer to the covalent bond that joins acarbohydrate molecule to another carbohydrate molecule. The terms“glucosidic linkage”, “glucosidic bond”, “linkage” and the like are usedinterchangeably herein and refer to a glycosidic linkage between twoglucose molecules. The term “alpha-1,6-glucosidic linkage” as usedherein refers to the covalent bond that joins alpha-D-glucose moleculesto each other through carbons 1 and 6 on adjacent alpha-D-glucose rings.This definition similarly applies to the terms “alpha-1,3-glucosidiclinkage”, “alpha-1,2-glucosidic linkage” and “alpha-1,4-glucosidiclinkage”, but using respective carbon numbers. Herein, “alpha-D-glucose”is referred to as “glucose” or “monomer”. All glucosidic linkagesdisclosed herein are alpha-glucosidic linkages, except as otherwisenoted.

The terms “poly alpha-1,3-glucan”, “alpha-1,3-glucan polymer”,“alpha-1,3-glucan” and the like are used interchangeably herein. Polyalpha-1,3-glucan comprises at least about 50% (e.g., ≥95%) alpha-1,3linkages in certain aspects.

The terms “dextran”, “dextran polymer”, “dextran molecule” and the likeare used interchangeably herein and refer to an alpha-glucan generallycomprising a main chain with substantially (mostly) alpha-1,6-linkedglucose monomers, typically with periodic branches linked to the mainchain by alpha-1,3, alpha-1,2, and/or alpha-1,4 linkages.

A dextran main chain comprises more than about 90-95% of all the glucosemonomers of a dextran polymer in some aspects. A dextran main chain insome instances can comprise substantially (or mostly) alpha-1,6linkages, meaning that it can have at least about 98.0% alpha-1,6linkages. A dextran main chain can comprise a small amount of alpha-1,3linkages in some aspects, meaning that it can have less than about 2.0%alpha-1,3 linkages.

Dextran branches typically are short, being one (pendant) to threeglucose monomers in length, and comprise less than about 10% of all theglucose monomers of a dextran polymer. Such short branches can comprisealpha-1,2, alpha-1,3, and/or alpha-1,4 linkages. Dextran in someembodiments can also have branches comprising mostly alpha-1,6 linkages;the length of such a branch can be similar to the length of the chainfrom which the branch originates.

The linkage profile of an alpha-glucan herein can be determined usingany method known in the art. For example, a linkage profile can bedetermined using methods that use nuclear magnetic resonance (NMR)spectroscopy (e.g., ¹³C NMR or ¹H NMR). These and other methods that canbe used are disclosed in Food Carbohydrates: Chemistry, PhysicalProperties, and Applications (S. W. Cui, Ed., Chapter 3, S. W. Cui,Structural Analysis of Polysaccharides, Taylor & Francis Group LLC, BocaRaton, FL, 2005), which is incorporated herein by reference.

“Molecular weight” herein can be represented as number-average molecularweight (Mn) or as weight-average molecular weight (Mw), the units ofwhich are in Daltons or grams/mole. Alternatively, molecular weight canbe represented as DPw (weight average degree of polymerization) or DPn(number average degree of polymerization). The molecular weight ofsmaller alpha-glucan polymers typically can be provided as “DP” (degreeof polymerization), which simply refers to the number of glucosescomprised within the alpha-glucan. Various means are known in the artfor calculating these molecular weight measurements, such as withhigh-pressure liquid chromatography (HPLC), size exclusionchromatography (SEC), or gel permeation chromatography (GPC).

The terms “glucosyltransferase enzyme”, “GTF enzyme”, “GTF”,“glucansucrase” and the like are used interchangeably herein. Theactivity of a glucosyltransferase herein catalyzes the reaction of thesubstrate sucrose to make the products alpha-glucan and fructose.Byproducts of a glucosyltransferase reaction can include glucose,various soluble gluco-oligosaccharides (DP2-DP7), and leucrose. Wildtype forms of glucosyltransferase enzymes generally contain (in theN-terminal to C-terminal direction) a signal peptide, a variable domain,a catalytic domain, and a glucan-binding domain. A glucosyltransferaseherein is classified under the glycoside hydrolase family 70 (GH70)according to the CAZy (Carbohydrate-Active EnZymes) database (Cantarelet al., Nucleic Acids Res. 37:D233-238, 2009). The term “dextransucrase”can optionally be used to characterize a glucosyltransferase enzyme thatproduces dextran.

The terms “enzymatic reaction” “glucosyltransferase reaction”, “glucansynthesis reaction” and the like are used interchangeably herein andtypically refer to a reaction that initially comprises at least water,sucrose, dextran and a glucosyltransferase enzyme. Such a reactionproduces graft copolymer, which can then be crosslinked as presentlydisclosed.

The term “absorb” and like terms as used herein refers to the action oftaking up (soaking up) an aqueous liquid. Absorption by a composition aspresently disclosed can be measured in terms of water retention value(WRV) and/or centrifugal retention capacity (CRC) as disclosed herein,for example.

The terms “aqueous liquid”, “aqueous fluid” and the like as used hereincan refer to water or an aqueous solution. An “aqueous solution” hereincan comprise one or more dissolved salts, where the maximal total saltconcentration can be about 3.5 wt% in some embodiments. Although aqueousliquids herein typically comprise water as the only solvent in theliquid, an aqueous liquid can optionally comprise one or more othersolvents (e.g., polar organic solvent) that are miscible in water. Thus,an aqueous solution can comprise a solvent having at least about 10 wt%water.

The term “household care product” and like terms typically refer toproducts, goods and services relating to the treatment, cleaning,caring, and/or conditioning of the home and its contents.

The term “personal care product” and like terms typically refer toproducts, goods and services relating to the treatment, cleaning,cleansing, caring, and/or conditioning of the person.

The term “medical product” and like terms typically refer to products,goods and services relating to the diagnosis, treatment, and/or care ofpatients.

The term “industrial product” and like terms typically refer toproducts, goods and services used in industrial settings, but not byindividual consumers.

The terms “percent by volume”, “volume percent”, “vol %”, “v/v %” andthe like are used interchangeably herein. The percent by volume of asolute in a solution can be determined using the formula: [(volume ofsolute)/(volume of solution)] × 100%.

The terms “percent by weight”, “weight percentage (wt%)”, “weight-weightpercentage (% w/w)” and the like are used interchangeably herein.Percent by weight refers to the percentage of a material on a mass basisas it is comprised in a composition, mixture, or solution.

The terms “sequence identity”, “identity” and the like as used hereinwith respect to a polypeptide amino acid sequence are as defined anddetermined in U.S. Pat. Appl. Publ. No. 2017/0002336, which isincorporated herein by reference.

A crosslinked graft copolymer or crosslinked dextran herein (and areaction for synthesis thereof) can optionally be characterized as being“isolated”, since it is synthetic/man-made, and/or has properties thatare not naturally occurring.

Unless otherwise disclosed, the term “increased” as used herein canrefer to a quantity or activity that is at least about 1 %, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,20%, 50%, 100%, or 200% more than the quantity or activity for which theincreased quantity or activity is being compared. The terms “increased”,“elevated”, “enhanced”, “greater than”, “improved” and the like are usedinterchangeably herein.

Further development of polysaccharide-based compositions is desired foracquiring enhanced superabsorption function. Thus, to address this need,disclosed herein are, for example, dextran-alpha-1,3-glucan graftcopolymers that, after enzymatic synthesis thereof, have beencrosslinked. Various embodiments of such crosslinked graft copolymershave enhanced aqueous liquid absorption characteristics.

Certain embodiments of the present disclosure concern a compositioncomprising a crosslinked graft copolymer, wherein the graft copolymerportion of the crosslinked graft copolymer comprises:

-   (i) a backbone comprising dextran, and-   (ii) poly alpha-1,3-glucan side chains comprising at least about 50%    alpha-1,3-glucosidic linkages.

In typical embodiments, one or more crosslinks of a crosslinked graftcopolymer are covalent (i.e., the graft copolymers are chemicallycrosslinked with each other). However, it is contemplated that one ormore crosslinks can be non-covalent in some alternative embodiments.Crosslinks herein can be between at least two graft copolymer molecules(i.e., intermolecular crosslinks). It is contemplated that crosslinks insome embodiments can also be intramolecular, such as between separatepoly alpha-1,3-glucan side chains of the same graft copolymer molecule,and/or between different sections of the dextran backbone of the samegraft copolymer molecule.

A crosslink herein typically joins moieties via two or more covalentbonds. Such a crosslink can comprise, for example, at least a covalentbond to an oxygen atom (previously of a hydroxyl group beforecrosslinking) of a glucose monomer, and a covalent bond to an oxygenatom (previously of a hydroxyl group before crosslinking) of anotherglucose monomer. A crosslink joining moieties via two covalent bonds canhave an atom (“crosslinking atom”) that is bonded to (i) an oxygen atomof a glucose monomer, and (ii) an oxygen atom of another glucosemonomer. A crosslinking atom(s) can optionally have one or more otherbonds to other atom(s) (e.g., hydrogen, oxygen) that typically arederived from the crosslinking agent used to create the crosslink. Forexample, if phosphoryl chloride (POCl₃, also known as phosphorusoxychloride) or sodium trimetaphosphate (STMP) is used to create acrosslink, such a crosslink can optionally be characterized as having aphosphorus atom as a single crosslinking atom; aside from its twocovalent bonds to oxygens of the glucose monomers being linked, thephosphorus atom is also bonded to an oxygen via a double-bond andanother oxygen via a single bond. A crosslinker in some embodiments canhave two or more (e.g., 3, 4, 5, 6, 7, 8 or more) crosslinking atoms;the number of covalent bonds that effectively link moieties in theseembodiments increases accordingly with the number of crosslinking atoms.

One or more crosslinks of a crosslinked graft copolymer can comprisephosphorus in some aspects of the present disclosure. An example of sucha crosslink is a phosphodiester bond. A phosphodiester bond hereintypically is formed between hydroxyl groups of glucose monomers. Forexample, a phosphodiester bond can be formed between a hydroxyl group ofa glucose monomer within a first graft copolymer and a hydroxyl group ofa glucose monomer within a second graft copolymer (such linkage isintermolecular in this example). A crosslinking agent that can be usedherein to prepare a crosslink comprising a phosphodiester bond can bePOCl₃, for example. In some aspects, a crosslinking agent that can beused to prepare a crosslink comprising phosphorus can include POCl₃,polyphosphate, or STMP.

As described above, a crosslink herein can be prepared using POCl₃,polyphosphate, or STMP as a crosslinking agent, for example. Otherexamples of suitable crosslinking agents include boron-containingcompounds (e.g., boric acid, diborates, tetraborates such as tetraboratedecahydrate, pentaborates, polymeric compounds such as Polybor®, alkaliborates), polyvalent metals (e.g., titanium-containing compounds such astitanium ammonium lactate, titanium triethanolamine, titaniumacetylacetonate, or polyhydroxy complexes of titanium;zirconium-containing compounds such as zirconium lactate, zirconiumcarbonate, zirconium acetylacetonate, zirconium triethanolamine,zirconium diisopropylamine lactate, or polyhydroxy complexes ofzirconium), glyoxal, glutaraldehyde, divinyl sulfone, epichlorohydrin,polycarboxylic acids (e.g., citric acid, malic acid, tartaric acid,succinic acid, glutaric acid, adipic acid), dichloro acetic acid, andpolyamines. Still other examples of suitable crosslinking agents aredescribed in U.S. Pat. Nos. 4462917, 4464270, 4477360 and 4799550, andU.S. Pat. Appl. Publ. No. 2008/0112907, which are all incorporatedherein by reference. A crosslinker in certain aspects can dissolve in anaqueous solvent herein. Yet in some aspects, a crosslinking agent is nota boron-containing compound (e.g., as described above).

A crosslink in certain aspects herein can involve (e.g., be preparedfrom) a carboxyl group that may have been derivatized onto a glucosemonomer. A graft copolymer can in certain aspects comprise addedcarboxyl groups for utilization in such crosslinking chemistry. Yet, insome aspects, a crosslinked graft copolymer does not comprise acrosslink based on this chemistry.

A crosslinked graft copolymer can in some aspects be surface-crosslinkedfollowing initial crosslinking (crosslinked at the polymer surface).Examples of surface-crosslinking schemes herein include using apolyhydroxyl compound (e.g., polyvinyl alcohol) and/or usingcarboxymethyl cellulose (CMC) plus a crosslinker (e.g., epichlorohydrin,STMP, phosphoric acid, aminopropyl siloxanes). Surface-crosslinking canoptionally involve (e.g., be prepared from), for example, a carboxylgroup that may have been derivatized onto a glucose monomer and/or acarboxyl group that may have been introduced during the initialcrosslinking. Surface-crosslinking herein can incorporate an agentand/or process as disclosed in any of U.S. Pat. Nos. 5462972, 6821331,7871640, 8361926, or 8486855, which are all incorporated herein byreference. Yet, in some aspects, a crosslinked graft copolymer is notsurface-crosslinked.

Aside from any effects from crosslinking itself, a crosslinked graftcopolymer typically has not been derivatized (e.g., not etherified,esterified, oxidized), nor has a graft copolymer (used to produce thecrosslinked graft copolymer) typically been derivatized.

A crosslinked graft copolymer herein can comprise a homogenous orheterogenous graft copolymer component. A crosslinked graft copolymerwith a homogenous graft copolymer component can be prepared using oneform, lot, or preparation of graft copolymer, for example, such as thatmade in a particular enzymatic reaction. A crosslinked graft copolymerwith a heterogenous graft copolymer component typically is preparedusing two or more different forms, lots, or preparations of graftcopolymer, for example, such as ones made in different enzymaticreactions. For example, graft copolymers respectively comprising about60 wt% dextran or 90 wt% dextran could be crosslinked to form acrosslinked graft copolymer with a heterogenous graft copolymercomponent.

A crosslinked graft copolymer in some embodiments can further comprisepoly alpha-1,3 glucan homopolymer that is not glucosidically linked to adextran backbone. Such embodiments can result from the co-production offree, non-primed poly alpha-1,3-glucan during enzymatic synthesis of adextran/poly-alpha-1,3-glucan graft copolymer (the latter of whichresults from alpha-1,3-glucan synthesis off of dextran primer). Suchfree poly alpha-1,3 glucan homopolymer can be chemically crosslinkedwithin these embodiments (e.g., crosslinked with graft copolymer), andcan be of any Mw as disclosed herein for poly alpha-1,3 glucan sidechains, for example.

A crosslinked graft copolymer as presently disclosed is typicallyinsoluble under aqueous conditions (aqueous insoluble). For example, acrosslinked graft copolymer can be insoluble or not completely dissolvedin water or another aqueous composition at a temperature up to about 50,60, 70, 80, 90, 100, 110, or 120° C. An aqueous composition herein suchas an aqueous solution can comprise a solvent having at least about 10wt% water. In some embodiments, a solvent is at least about 20, 30, 40,50, 60, 70, 80, 90, or 100 wt% water (or any integer value between 10and 100 wt%), for example. In some embodiments, the pH of an aqueoussolution is between 4 and 9.

The degree of crosslinking of a crosslinked graft copolymer in someaspects can be determined using the following formula (expressed as apercentage): [(total number of reactive groups in crosslinking agentused) / (total number of disaccharide units in the graft copolymermolecules)] × 100. The degree of crosslinking in some aspects iscontemplated to be between about 0.5%-70%, 0.5%-50%, 2.5%-70%, 2.5%-50%,5%-70%, or 5%-50%, for example. The degree of crosslinking can bemodified accordingly by altering the level of crosslinking agent used,for example.

A dextran forming the backbone of a graft copolymer portion of acrosslinked graft copolymer herein can comprise, for example, about, orat least about, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%alpha-1,6-glucosidic linkages. Such a percent alpha-1,6 linkage profiletakes into account the total of all linkages in the dextran (main chainand branch portions combined). “Dextran branches” and like terms hereinare meant to encompass any branches that exist in a dextran polymerprior to its use to prepare a graft copolymer. In some embodiments, adextran comprises a main chain comprising about, or at least about, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%alpha-1,6-glucosidic linkages. In some embodiments, the dextran iscompletely linear (100% alpha-1,6-glucosidic linkages).

A dextran herein can comprise, for example, about, or at least about,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, or 20% alpha-1,4, alpha-1,3 and/or alpha-1,2 glucosidiclinkages. Typically, such linkages exist entirely, or almost entirely,in branch portions of the dextran, including branch points. In someembodiments, dextran branches may comprise one, two (e.g., alpha-1,4 andalpha-1,3; alpha-1,4 and alpha-1,2; alpha-1,3 and alpha-1,2), or allthree of these types of linkages. The total percentage of alpha-1,4,alpha-1,3 and/or alpha-1,2 glucosidic linkages in a dextran herein istypically not greater than 50%. In some aspects, such as with dextrancomprising a main chain having about, or at least about, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% alpha-1,6-glucosidiclinkages, such dextran comprises about, or at least about, or less thanabout, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% alpha-1,4, alpha-1,3and/or alpha-1,2 glucosidic linkages.

A branch point of a dextran herein can comprise an alpha-1,4, alpha-1,3,or alpha-1,2 glucosidic linkage (e.g., a branch may be alpha-1,3-linkedto a dextran main chain). In some embodiments, all three of these branchpoints may exist, whereas in some embodiments only one or two (e.g.,alpha-1,4 and alpha-1,3; alpha-1,4 and alpha-1,2; alpha-1,3 andalpha-1,2) types of these branch points exist. It is contemplated that abranch point occurs on average every (or at least about every) 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 10 to 30, 15 to 25, 20 to 30, or 20 to40 glucose units of a dextran main chain, for example. Branches of adextran molecule comprising alpha-1,4, alpha-1,3, and/or alpha-1,2glucosidic linkages herein typically are one to three glucose monomersin length and comprise less than about 5-10% of all the glucose monomersof a dextran polymer. A branch comprising one glucose unit can beoptionally be referred to as a pendant glucose group. In someembodiments, the branches of a dextran molecule can comprise about, orless than about, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of all theglucose monomers of a dextran molecule. A dextran in certain embodimentscan have about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% branch pointsas a percent of the glucosidic linkages in the polymer. The glucosidiclinkage profile of a branch herein can optionally be characterized toinclude the glucosidic linkage by which the branch is linked to anotherchain.

A backbone of a graft copolymer in certain embodiments can be comprisedentirely of a dextran as presently disclosed. However, in some aspects,a backbone can comprise other elements. For example, a graft copolymerbackbone can comprise poly alpha-1,3-glucan originating from thenon-reducing side of a dextran main chain, by virtue of the main chain(at its non-reducing end) serving to prime poly alpha-1,3-glucansynthesis during synthesis of the graft copolymer.

The molecular weight (Mw [weight-average molecular weight]) of a dextranherein can be about, or at least about, or less than about, 1000, 2000,5000, 10000, 25000, 40000, 50000, 75000, 100000, 125000, 150000, 175000,200000, 240000, 250000, 500000, 750000, or 1000000 Daltons, or be in arange of about 100000-200000, 125000-175000, 130000-170000,135000-165000, 140000-160000, or 145000-155000 Daltons, for example. Insome aspects, dextran can have a Mw of about, or at least about, or lessthan about, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 millionDaltons, or which is in a range of about 10-80, 20-70, 30-60, 40-50,50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 110-200, 120-200,50-180, 60-180, 70-180, 80-180, 90-180, 100-180, 110-180, 120-180,50-160, 60-160, 70-160, 80-160, 90-160, 100-160, 110-160, 120-160,50-140, 60-140, 70-140, 80-140, 90-140, 100-140, 110-140, 120-140,50-120, 60-120, 70-120, 80-120, 90-120, 90-110, 100-120, 110-120,50-110, 60-110, 70-110, 80-110, 90-110, 100-110, 50-100, 60-100, 70-100,80-100, 90-100, or 95-105 million Daltons. Dextran with a Mw of at leastabout 50 million Daltons (e.g., 50-200 million Daltons) herein canoptionally be referred to as a “very large dextran” or “very highmolecular weight dextran”. The Mw of dextran in some aspects is notbelow 100000 Daltons, and thus is not T10 (Mw = 10000), T25 (Mw =25000), or T40 (Mw = 40000) dextran, for example. Any dextran Mw hereincan optionally be expressed as weight-average degree of polymerization(DPw), which is Mw divided by 162.14.

A very large dextran in some aspects can comprise (i) about 87-93 wt%glucose linked only at positions 1 and 6; (ii) about 0.1-1.2 wt% glucoselinked only at positions 1 and 3; (iii) about 0.1-0.7 wt% glucose linkedonly at positions 1 and 4; (iv) about 7.7-8.6 wt% glucose linked only atpositions 1, 3 and 6; and (v) about 0.4-1.7 wt% glucose linked only at:(a) positions 1, 2 and 6, or (b) positions 1, 4 and 6. In certainembodiments, a dextran can comprise (i) about 89.5-90.5 wt% glucoselinked only at positions 1 and 6; (ii) about 0.4-0.9 wt% glucose linkedonly at positions 1 and 3; (iii) about 0.3-0.5 wt% glucose linked onlyat positions 1 and 4; (iv) about 8.0-8.3 wt% glucose linked only atpositions 1, 3 and 6; and (v) about 0.7-1.4 wt% glucose linked only at:(a) positions 1, 2 and 6, or (b) positions 1, 4 and 6. Suitable examplesof very large dextran herein are described in Examples 5 and 6 below.

A very large dextran in some aspects can comprise about 87, 87.5, 88,88.5, 89, 89.5, 90, 90,5, 91, 91.5, 92, 92.5, or 93 wt% glucose linkedonly at positions 1 and 6. There can be about 87-92.5, 87-92, 87-91.5,87-91, 87-90.5, 87-90, 87.5-92.5, 87.5-92, 87.5-91.5, 87.5-91,87.5-90.5, 87.5-90, 88-92.5, 88-92, 88-91.5, 88-91, 88-90.5, 88-90,88.5-92.5, 88.5-92, 88.5-91.5, 88.5-91, 88.5-90.5, 88.5-90, 89-92.5,89-92, 89-91.5, 89-91, 89-90.5, 89-90, 89.5-92.5, 89.5-92, 89.5-91.5,89.5-91, or 89.5-90.5 wt% glucose linked only at positions 1 and 6, insome instances.

A very large dextran in some aspects can comprise about 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, or 1.2 wt% glucose linked onlyat positions 1 and 3. There can be about 0.1-1.2, 0.1-1.0, 0.1-0.8,0.3-1.2, 0.3-1.0, 0.3-0.8, 0.4-1.2, 0.4-1.0, 0.4-0.8, 0.5-1.2, 0.5-1.0,0.5-0.8, 0.6-1.2, 0.6-1.0, or 0.6-0.8 wt% glucose linked only atpositions 1 and 3, in some instances.

A very large dextran in some aspects can comprise about 0.1, 0.2, 0.3,0.4, 0.5, 0.6, or 0.7 wt% glucose linked only at positions 1 and 4.There can be about 0.1-0.7, 0.1-0.6, 0.1-0.5, 0.1-0.4, 0.2-0.7, 0.2-0.6,0.2-0.5, 0.2-0.4, 0.3-0.7, 0.3-0.6, 0.3-0.5, or 0.3-0.4 wt% glucoselinked only at positions 1 and 4, in some instances.

A very large dextran in some aspects can comprise about 7.7, 7.8, 7.9,8.0, 8.1, 8.2, 8.3, 8.4, 8.5, or 8.6 wt% glucose linked only atpositions 1, 3 and 6. There can be about 7.7-8.6, 7.7-8.5, 7.7-8.4,7.7-8.3, 7.7-8.2, 7.8-8.6, 7.8-8.5, 7.8-8.4, 7.8-8.3, 7.8-8.2, 7.9-8.6,7.9-8.5, 7.9-8.4, 7.9-8.3, 7.9-8.2, 8.0-8.6, 8.0-8.5, 8.0-8.4, 8.0-8.3,8.0-8.2, 8.1-8.6, 8.1-8.5, 8.1-8.1, 8.1-8.3, or 8.1-8.2 wt% glucoselinked only at positions 1, 3 and 6, in some instances.

A very large dextran in some aspects can comprise about 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, or 1.7 wt% glucoselinked only at (a) positions 1, 2 and 6, or (b) positions 1, 4 and 6.There can be about 0.4-1.7, 0.4-1.6, 0.4-1.5, 0.4-1.4, 0.4-1.3, 0.5-1.7,0.5-1.6, 0.5-1.5, 0.5-1.4, 0.5-1.3, 0.6-1.7, 0.6-1.6, 0.6-1.5, 0.6-1.4,0.6-1.3, 0.7-1.7, 0.7-1.6, 0.7-1.5, 0.7-1.4, 0.7-1.3, 0.8-1.7, 0.8-1.6,0.8-1.5, 0.8-1.4, 0.8-1.3 wt% glucose linked only at (a) positions 1, 2and 6, or (b) positions 1, 4 and 6, in some instances.

“Glucose (glucose monomers) linked at positions 1 and 6” herein refersto a glucose monomer of dextran in which only carbons 1 and 6 of theglucose monomer are involved in respective glucosidic linkages with twoadjacent glucose monomers. This definition likewise applies to glucose(i) “linked at positions 1 and 3”, and (ii) “linked at positions 1 and4”, taking into account, accordingly, the different carbon positionsinvolved in each respective linkage. “Glucose (glucose monomers) linkedat positions 1, 3 and 6” herein refers to a glucose monomer of dextranin which carbons 1, 3 and 6 of the glucose monomer are involved inrespective glucosidic linkages with three adjacent glucose monomers. Aglucose linked only at positions 1, 3 and 6 is a branch point. Thisdefinition likewise applies to glucose linked at (i) positions 1, 2 and6, and (ii) positions 1, 4 and 6, but taking into account, accordingly,the different carbon positions involved in each respective linkage.Glucose positions (glucose carbon positions) 1, 2, 3, 4 and 6 herein areas known in the art (depicted in the following structure):

The glucosidic linkage profile of a very large dextran can be determinedusing dextran produced following any protocol disclosed herein. Anexample of a suitable linkage determination protocol can be similar to,or the same as, the protocols disclosed in U.S. Appl. Publ. No.2016/0122445 (e.g., para. 97 or Example 9 therein), which isincorporated herein by reference.

It is believed that very large dextran herein can be a branchedstructure in which there are long chains (containing mostly or allalpha-1,6-linkages) that iteratively branch from each other (e.g., along chain can be a branch from another long chain, which in turn canitself be a branch from another long chain, and so on). The branchedstructure may also comprise short branches from the long chains; theseshort chains are believed to mostly comprise alpha-1,3 and -1,4linkages, for example. Branch points in the very large dextran, whetherfrom a long chain branching from another long chain, or a short chainbranching from a long chain, appear to comprise alpha-1,3, -1,4, or -1,2linkages off of a glucose involved in alpha-1,6 linkage. On average,about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 15-35%,15-30%, 15-25%, 15-20%, 20-35%, 20-30%, 20-25%, 25-35%, or 25-30% of allbranch points of very large dextran in some embodiments branch into longchains. Most (>98% or 99%) or all the other branch points branch intoshort chains.

The long chains of a very large dextran branching structure can besimilar in length in some aspects. By being similar in length, it ismeant that the individual length (DP) of at least 70%, 75%, 80%, 85%, or90% of all the long chains in a branching structure is within plus/minus15% (or 10%, 5%) of the mean length of all the long chains of thebranching structure. In some aspects, the mean length (average length)of the long chains of a very large dextran is about 10-50 DP (i.e.,10-50 glucose monomers). For example, the mean individual length of thelong chains can be about 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,30, 35, 40, 45, 50, 10-50, 10-40, 10-30, 10-25, 10-20, 15-50, 15-40,15-30, 15-25, 15-20, 20-50, 20-40, 20-30, or 20-25 DP.

Long chains in certain embodiments of very large dextran can comprisesubstantially alpha-1,6-glucosidic linkages and a small amount (lessthan 2.0%) of alpha-1,3- and/or alpha-1,4-glucosidic linkages. Forexample, very large dextran long chains can comprise about, or at leastabout, 98%, 98.25%, 98.5%, 98.75%, 99%, 99.25%, 99.5%, 99.75%, or 99.9%alpha-1,6-glucosidic linkages. A dextran long chain in certainembodiments does not comprise alpha-1,4-glucosidic linkages (i.e., sucha long chain has mostly alpha-1,6 linkages and a small amount ofalpha-1,3 linkages). Conversely, a dextran long chain in someembodiments does not comprise alpha-1,3-glucosidic linkages (i.e., sucha long chain has mostly alpha-1,6 linkages and a small amount ofalpha-1,4 linkages). Any dextran long chain of the above embodiments mayfurther not comprise alpha-1,2-glucosidic linkages, for example. Stillin some aspects, a dextran long chain can comprise 100%alpha-1,6-glucosidic linkages (excepting the linkage used by such longchain to branch from another chain).

Short chains of a very large dextran molecule in some aspects are one tothree glucose monomers in length and comprise less than about 5-10% ofall the glucose monomers of the dextran polymer. At least about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or all of, short chainsherein are 1-3 glucose monomers in length. The short chains of a dextranmolecule can comprise less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, or 1% of all the glucose monomers of a very large dextran molecule,for example.

Short chains of a very large dextran molecule in some aspects cancomprise alpha-1,2-, alpha-1,3-, and/or alpha-1,4-glucosidic linkages.Short chains, when considered all together (not individually) maycomprise (i) all three of these linkages, or (ii) alpha-1,3- andalpha-1,4-glucosidic linkages, for example.

Regarding a graft copolymer comprising very large dextran, it iscontemplated that a “backbone” herein is a long chain of the very largedextran. A poly alpha-1,3-glucan side chain can be linked to a longchain of a very large dextran in a manner as presently disclosedthroughout (e.g., extension from the non-reducing end of a short chain[e.g., pendant glucose] or of a long chain).

The Mw of a very large dextran herein is about 50-200 million, or any Mwas disclosed above for dextran falling within this range.

The z-average radius of gyration of a very large dextran herein can beabout 200-280 nm. For example, the z-average Rg can be about 200, 205,210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, or280 nm (or any integer between 200-280 nm). As other examples, thez-average Rg can be about 200-280, 200-270, 200-260, 200-250, 200-240,200-230, 220-280, 220-270, 220-260, 220-250, 220-240, 220-230, 230-280,230-270, 230-260, 230-250, 230-240, 240-280, 240-270, 240-260, 240-250,250-280, 250-270, or 250-260 nm.

The term “radius of gyration” (Rg) herein refers to the mean radius ofdextran, and is calculated as the root-mean-square distance of a dextranmolecule’s components (atoms) from the molecule’s center of gravity. Rgcan be provided in Angstrom or nanometer (nm) units, for example. The“z-average radius of gyration” of dextran herein refers to the Rg ofdextran as measured using light scattering (e.g., MALS). Methods formeasuring z-average Rg are known and can be used herein, accordingly.For example, z-average Rg can be measured as disclosed in U.S. Pat. No.7531073, U.S. Pat. Appl. Publ. Nos. 2010/0003515 and 2009/0046274, Wyatt(Anal. Chim. Acta 272:1-40), and Mori and Barth (Size ExclusionChromatography, Springer-Verlag, Berlin, 1999), all of which areincorporated herein by reference.

The Mw and/or z-average Rg of very large dextran in some aspects can bemeasured following a protocol similar to, or the same as, the protocolsdisclosed in U.S. Appl. Publ. No. 2016/0122445 (e.g., para. 105 orExample 9 therein), which is incorporated herein by reference.

A very large dextran herein can be enzymatically synthesized accordingto the disclosure of U.S. Appl. Publ. No. 2016/0122445, for example,which is incorporated herein by reference. For example, as described inthis reference, such a dextran can be produced in a suitable reactioncomprising GTF 0768, or a GTF comprising an amino acid sequence that isat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto the amino acid sequence of GTF 0768.

A graft copolymer portion of a crosslinked graft copolymer hereincomprises a dextran backbone from which there are poly alpha-1,3-glucanside chains comprising at least about 50% alpha-1,3-glucosidic linkages.These side chains typically can be obtained via reacting a dextran aspresently disclosed herein with a glucosyltransferase that cansynthesize poly alpha-1,3-glucan. For clarity purposes, these sidechains ought not be considered branches of dextran.

A poly alpha-1,3-glucan side chain in certain aspects can compriseabout, or at least about, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 69%, 70%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%alpha-1,3 glucosidic linkages. A side chain is contemplated in someaspects to be synthesized with a glucosyltransferase enzyme using apendant glucose or other branch of dextran (both of which presentnon-reducing ends to the enzyme for extension) as a primer. Where a sidechain is synthesized from a pendant glucose that is itselfalpha-1,3-linked to the dextran main chain, the resulting side chain canhave 100% or a very high (e.g., 98% or greater) percentage ofalpha-1,3-glucosidic linkages. In some embodiments, the glucosidiclinkage between a dextran main chain and a pendant glucose or longerbranch is considered a linkage of the side chain. In some embodiments,the glucosidic linkage between a dextran main chain and a branch, aswell as the glucosidic linkages within a branch from which a side chainwas synthesized, are considered in determining the linkage profile ofthe side chain. In some alternative embodiments, a poly alpha-1,3-glucanside chain can comprise about, or at least about, 30% alpha-1,3glucosidic linkages. The balance of linkages in any polyalpha-1,3-glucan side chain herein typically can be with alpha-1,6linkages.

The Mw of a poly alpha-1,3-glucan side chain herein can be about, or atleast about 1620, 1650, 1700, 2000, 5000, 10000, 15000, 16200, 20000,25000, 30000, 40000, 50000, 60000, 70000, 75000, 80000, 90000, 100000,110000, 120000, 125000, 130000, 140000, 150000, 160000, 162000, or165000 Daltons, for example. It is contemplated that the side chains ofa graft copolymer herein are relatively homogenous in size. Forinstance, the sides chains of a graft copolymer can each have a Mw inthe range of about 150000-165000, 155000-165000, or 160000-165000Daltons. The average Mw of the side chains of a graft copolymer can alsobe referred to, if desired; any of the foregoing side chain Mw’s can beconsidered an average Mw of all the side chains of a copolymer. Any ofthe side chain Mw’s (or any glucan Mw) disclosed herein can optionallybe characterized in terms of DPw (i.e., Mw/162.14).

The number of poly alpha-1,3-glucan side chains of a graft copolymerherein can be, or can be at least, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30,for example. In some embodiments, the number of side chains is 4, 5, or6, for example. The foregoing number of poly alpha-1,3-glucan sidechains in some aspects is a characteristic of side chains that are atleast about 100000, 120000, 140000, 160000, 162000, or 165000 Daltons.Still, in further aspects, the foregoing number of poly alpha-1,3-glucanside chains can characterize a graft copolymer in which the dextrancomponent has a pendant glucose and/or branch (from which a side chaincan be primed/synthesized) on average once every 15, 16, 17, 18, 19, 20,21, 22, 23, 24, or 25 glucose units of a dextran main chain. Based onthe size of a dextran component (e.g., 100000-200000 Daltons), thepositioning of branches/pendant glucoses on the dextran main chain(e.g., about one every 20 glucose units), and the number of polyalpha-1,3-glucan side chains of a graft copolymer, it is contemplated insome cases that a graft copolymer has a majority (e.g., at least 80%,85%, 90%, 95%) of its original dextran branches/pendant glucosesnon-extended into a poly alpha-1,3-glucan side chain (i.e., most of thebranches/pendant glucoses are as they existed in the dextran before usethereof to synthesize a graft copolymer). Still, in some otherembodiments, it is believed possible that a graft copolymer herein canhave up to about 50, 100, 500, 1000, 5000, 10000, 15000, or 20000 polyalpha-1,3-glucan side chains.

The Mw of a graft copolymer portion of a crosslinked graft copolymerherein (i.e., the combined Mw of the original dextran molecule and thepoly alpha-1,3-glucan side chains of a graft copolymer) can be about, orat least about, 750000, 800000, 900000, 1000000, 1100000, 1200000,1300000, 1400000, 1500000, 1600000, 1700000, 1800000, 1900000, or2000000 Daltons, for example. The Mw of a graft copolymer that comprisesa very large dextran component in some embodiments is believed to besimilar to the weight as disclosed above for the very large dextrancomponent itself, but with the addition of about 0.5, 0.75, 1, 1.25,1.5, 1.75 or 2 million Daltons (in embodiments in which there are a fewpoly alpha-1,3-glucan side chains). The polydispersity index (Mw/Mn)(PDI) of a graft copolymer herein can be about, at least about, or lessthan about, 5.0, 4.75, 4.5, 4.25, 4.0, 3.75, 3.5, 3.25, 3.0, 2.75, 2.5,2.25, or 2.0, for example.

In certain embodiments, a graft copolymer can comprise about, or atleast about, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99, 50-95, 60-95, 50-90, or60-90 wt% one or more dextran compounds as disclosed herein.

A graft copolymer portion of a crosslinked graft copolymer herein can beproduced using an enzymatic reaction as disclosed in International Appl.Publ. No. WO2017/079595 (Appl. No. PCT/US2016/060579), which isincorporated herein by reference, for example. Such an enzymaticreaction typically comprises at least: (i) water, (ii) sucrose, (iii)one or more dextran compounds as disclosed herein, and (iv) aglucosyltransferase enzyme that synthesizes poly alpha-1,3-glucan. Polyalpha-1,3-glucan synthesis by a glucosyltransferase enzyme in thisreaction can, in part at least, be via use of the dextran as a primerfor poly alpha-1,3-glucan synthesis. Following enzymatic production ofdextran-poly alpha-1,3-glucan graft copolymer, it can be chemicallycrosslinked to produce a crosslinked graft copolymer as presentlydisclosed.

The initial concentration of dextran in an enzymatic reaction forpreparing graft copolymer herein can be about, or at least about, 0.5g/L, 1.0 g/L, 1.5 g/L, 2 g/L, 2.5 g/L, 3 g/L, 4 g/L, 5 g/L, 7.5 g/L, 10g/L, 15 g/L, 20 g/L, or 25 g/L, for example. “Initial concentration ofdextran” refers to the dextran concentration in a glucosyltransferasereaction just after all the reaction components have been added (e.g.,at least water, sucrose, dextran, glucosyltransferase enzyme). Dextranfor entry into a reaction can be from a commercial source or preparedenzymatically, for example. Dextran produced enzymatically (e.g., usingdextransucrase) can, in some aspects, be (i) isolated in some mannerfrom an initial dextran synthesis enzymatic reaction (e.g., separatedfrom a dextransucrase reaction) and then entered into an enzymaticreaction for alpha-1,3-glucan side chain synthesis, or (ii) entered intoan enzymatic reaction for alpha-1,3-glucan side chain synthesis withoutbeing separated from an initial dextran synthesis enzymatic reaction(e.g., completed and/or heat-killed reaction is used directly for thealpha-1,3-glucan side chain synthesis reaction).

An enzymatic reaction for producing a graft copolymer typicallycomprises a glucosyltransferase enzyme that can synthesize polyalpha-1,3-glucan comprising at least about 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% alpha-1,3-glucosidic linkages.Such an enzyme can synthesize poly alpha-1,3-side chains (as disclosedabove) from dextran primer sites, forming a dextran-polyalpha-1,3-glucan graft copolymer herein. In particular aspects, aglucosyltransferase enzyme can synthesize poly alpha-1,3-glucan that (i)comprises about 100%, or at least about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99%, alpha-1,3-glucosidic linkages, and/or (ii) is atleast about 16200 Daltons.

A glucosyltransferase enzyme in certain embodiments for producing polyalpha-1,3-glucan side chains can comprise, or consist of, an amino acidsequence as disclosed in U.S. Pat. Appl. Publ. No. 2014/0087431, forexample, which is incorporated herein by reference. Examples of suchsequences include those that are 100% identical to, or at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identicalto, SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 26, 28, 30, 34, or 59as disclosed in U.S. Pat. Appl. Publ. No. 2014/0087431, and haveglucosyltransferase activity. A glucosyltransferase enzyme with SEQ IDNO:2, 4, 8, 10, 14, 20, 26, 28, 30, or 34 can synthesize polyalpha-1,3-glucan comprising at least about 90% alpha-1,3-glucosidiclinkages in some aspects.

The temperature of an enzymatic reaction for producing a graft copolymercan be controlled, if desired. In certain embodiments, the temperatureof a reaction can be between about 5° C. to about 50° C., about 20° C.to about 40° C., or about 20° C. to about 30° C. (e.g., about 22-25°C.). The pH of an enzymatic reaction in certain embodiments can bebetween about 4.0 to about 8.0, or between about 5.0 to about 6.0.Alternatively, the pH can be about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0,7.5, or 8.0, for example. The pH can be adjusted or controlled by theaddition or incorporation of a suitable buffer, including but notlimited to: phosphate, tris, citrate, or a combination thereof. Bufferconcentration in a glucan synthesis reaction can be from 0 mM to about100 mM, or about 10, 20, or 50 mM, for example.

The initial concentration of sucrose in an enzymatic reaction forproducing a graft copolymer can be about 20-400, 200-400, 250-350,75-175, or 50-150 g/L, for example. In some aspects, the initialconcentration of sucrose can be about, or at least about, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 180, 200, 250, 300, or400 g/L, for example. “Initial concentration of sucrose” refers to thesucrose concentration in a glucosyltransferase reaction just after allthe reaction components have been added (e.g., at least water, sucrose,dextran, glucosyltransferase enzyme).

One or more glucosyltransferase enzymes may be used in an enzymaticreaction for producing a graft copolymer. An enzymatic reaction hereinmay contain one, two, or more glucosyltransferase enzymes, for example.In some aspects, only one or two glucosyltransferase enzymes is/arecomprised in a reaction. A reaction composition herein can be, andtypically is, cell-free (e.g., no whole cells present). A reactioncomposition can be contained within any vessel (e.g., an inertvessel/container) suitable for applying one or more reaction conditionsdisclosed herein. An inert vessel in some aspects can be of stainlesssteel, plastic, or glass (or comprise two or more of these components)and be of a size suitable to contain a particular reaction. Typically,the reaction time can be about 1, 2, 3, 4, 5, 10, 12, 24, 36, 48, 60,72, 84, or 96 hours.

Following its enzymatic synthesis, a graft copolymer can be isolated(e.g., by filtration or centrifugation), if desired, prior to beingcrosslinked. In doing so, the graft copolymer is separated from most ofthe reaction solution, which may comprise water, fructose, residualsucrose and certain byproducts (e.g., leucrose, soluble oligosaccharidesDP2-DP7, glucose). Isolation can optionally further comprise washing agraft copolymer product one, two, or more times with water or otheraqueous liquid, and/or drying the product. Such washing can use washvolumes of about, or at least about, 0.5-, 1-, 1.5-, or 2-times thevolume of the original reaction or of a product sample, and/or involvefiltration and/or centrifugation, for example.

A crosslinked graft copolymer as presently disclosed can be produced,for example, by contacting a graft copolymer herein with at least acrosslinking agent and a solvent. This process step can optionally becharacterized as contacting a graft copolymer with a crosslinking agentunder aqueous conditions or non-aqueous conditions, depending on thesolvent being used. Any crosslinking agent and/or graft copolymerdisclosed herein can be employed accordingly. Any process parameterdisclosed below and in the Examples can likewise be applied in theseproduct-by-process embodiments.

Further disclosed herein is a method/process of producing a crosslinkedgraft copolymer. This method can comprise:

-   (a) contacting at least a solvent, a crosslinking agent, and a graft    copolymer as presently disclosed, whereby a crosslinked graft    copolymer is produced, and-   (b) optionally, isolating the crosslinked graft copolymer produced    in step (a). Method step (a) can optionally be characterized as    contacting a graft copolymer with a crosslinking agent under aqueous    or non-aqueous conditions (depending on the solvent), and/or can    optionally be characterized as a crosslinking reaction. Any    crosslinking agent and/or graft copolymer disclosed herein can be    employed in this method accordingly. In the contacting step of the    above process and product-by-process embodiments, it is generally    desired that such is conducted under conditions suitable for    allowing the crosslinking agent to make a crosslink. It should be    evident from the present disclosure that a graft copolymer itself,    which is entered into a crosslinking reaction, typically is made    enzymatically as disclosed herein without any chemical crosslinking.

A crosslinking reaction herein can be performed under aqueous conditionsin certain aspects. For example, a reaction can be comprise, optionallyas a first step, providing a preparation (typically a slurry or mixture)of at least one graft copolymer (e.g., any as disclosed herein) in anaqueous liquid (e.g., water). The wt% of graft copolymer in such apreparation can be about, or at least about, 1, 5, 10, 15, 20, 25, 30,1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 5-30, 5-25, 5-20, 5-15, 5-10, 10-30,10-25, 10-20, or 10-15, for example (such a wt% can likewise be appliedto a non-aqueous reaction, if desired). This preparation can optionallybe incubated, preferably with agitation, for about, or at least about,0.25, 0.50, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, or 48 hours,and/or be at room temperature of a temperature of about 15, 20, 25, 30,35, 40, 50, 15-25, 15-30, 15-40, 15-50, 20-25, 20-30, 20-40, or 20-50°C. This preparation typically is made first without pH adjustment, butcan optionally be prepared simultaneously with pH adjustment (below).

The pH of the aqueous preparation can in certain aspects be adjusted(increased or decreased) accordingly. For example, such as when usingPOCl₃ as a crosslinking agent, a base (e.g., sodium hydroxide [NaOH])can be added to raise the pH to about 8, 8.5, 9, 9.5, 10, 10.5, 11,11.5, 12, 8-12, 9-12, 10-12, 8-11.5, 9-11.5, or 10-11.5. A pH-adjustedpreparation can optionally be incubated, preferably with agitation, forat least about 10, 15, 20, 25, 30, 45 or 60 minutes, and/or be at atemperature as listed above. Adjustment of pH is generally done before,but can optionally be done simultaneously with, addition of acrosslinking agent (below). Increasing pH in some aspects can partiallyor completely dissolve a graft copolymer.

A crosslinking agent (e.g., any as disclosed herein that can dissolve inaqueous conditions) is dissolved in the preparation, typically followingpH-adjustment. The concentration of the crosslinking agent in theresulting preparation can be about, or at least about, 0.2, 0.4, 0.5, 1,1.5, 1.6, 1.7, 2, 4, 6, 8, 10, 0.5-2, 1-2, 1.5-2, or 1.5-1.7 wt%, forexample (such a wt% can likewise be applied to a non-aqueous reaction,if desired). Agitation (e.g., shaking or stirring) is typically appliedwhile dissolving the crosslinking agent. This preparation is typicallyincubated, preferably with agitation, for at least about 0.25, 0.50, 1,2, 3, 4, or 5 hours, and/or be at a temperature as listed above.

A crosslinking reaction, if pH-adjusted, can optionally be neutralizedupon completion (e.g., using HCl if pH had been increased), orneutralized while isolating the crosslinked graft copolymer product ofthe reaction. Neutralization typically brings a pH around 7.0 (e.g.,6.0-8.0, 6.5-7.5, 6.8-7.2).

The aforementioned conditions/parameters for performing a crosslinkingreaction can be adjusted accordingly, depending on the type ofcrosslinker being employed, for example.

A crosslinked graft copolymer produced in a crosslinking reaction hereincan optionally be isolated. For example, a crosslinked product can beseparated by filtration or centrifugation (or any other method known inthe art for removal of liquids from solids) from thereaction/post-reaction liquid. Isolation can optionally further comprisewashing a crosslinked product one, two, or more times with water orother aqueous liquid, and/or drying the product. Washing in some aspectscan be done such that no salts (e.g., NaCl) can be detected in thewashed product. Drying in some aspects can be performed using any methodknown in the art, such as vacuum drying, air drying, or freeze drying.Drying can optionally be performed at a temperature of at least about70, 80, 90, or 70-90° C. Dried product can be made into a particulateform, if desired, such as through crushing and/or grinding.

The percent yield of a crosslinked graft copolymer product of acrosslinking reaction herein can be about, or at least about, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%,for example. Percent yield of a crosslinked graft copolymer can bemeasured, for instance, by dividing the actual product yield by thetheoretical product yield, and multiplying by 100%.

A crosslinked graft copolymer as comprised in a composition herein canabsorb an aqueous liquid. An aqueous liquid can be water for instance.An aqueous liquid in certain aspects can be an aqueous solution, such asa salt solution (saline solution). A salt solution can optionallycomprise about, or at least about, 0.01, 0.025, 0.05, 0.075, 0.1, 0.25,0.5, 0.75, 0.9, 1.0, 1.25, 1.5, 1.75, 2.0, 2.5, 3.0, 0.5-1.5, 0.5-1.25,0.5-1.0, 0.75-1.5, 0.75-1.25, or 0.75-1.0 wt% of salt (such wt% valuestypically refer to the total concentration of one or more salts).Examples of a salt that can be used in an aqueous solution hereininclude one or more sodium salts (e.g., NaCl, Na₂SO₄). Othernon-limiting examples of salts include those having (i) an aluminum,ammonium, barium, calcium, chromium (II or III), copper (I or II), iron(II or III), hydrogen, lead (II), lithium, magnesium, manganese (II orIII), mercury (I or II), potassium, silver, sodium strontium, tin (II orIV), or zinc cation, and (ii) an acetate, borate, bromate, bromide,carbonate, chlorate, chloride, chlorite, chromate, cyanamide, cyanide,dichromate, dihydrogen phosphate, ferricyanide, ferrocyanide, fluoride,hydrogen carbonate, hydrogen phosphate, hydrogen sulfate, hydrogensulfide, hydrogen sulfite, hydride, hydroxide, hypochlorite, iodate,iodide, nitrate, nitride, nitrite, oxalate, oxide, perchlorate,permanganate, peroxide, phosphate, phosphide, phosphite, silicate,stannate, stannite, sulfate, sulfide, sulfite, tartrate, or thiocyanateanion. Thus, any salt having a cation from (i) above and an anion from(ii) above can be in an aqueous liquid as presently disclosed, forexample.

Absorption of an aqueous liquid by a crosslinked graft copolymer ascomprised in a composition herein can be gauged by measuring the waterretention value (WRV) of the crosslinked graft copolymer, for example.WRV herein can be measured by any means known in the art, such as viathe methodology disclosed in U.S. Pat. Appl. Publ. No. 2016/0175811(e.g., Example 7 therein), which is incorporated herein by reference.Briefly, WRV can be calculated using the following formula: ((mass ofwet crosslinked graft copolymer - mass of dry crosslinked graftcopolymer) / mass of dry crosslinked graft copolymer) * 100. WRV can bemeasured with respect to any aqueous liquid as presently disclosed, forexample. Thus, while the term WRV contains the word “water”, it would beunderstood that WRV can be measured with regard to any type of aqueousliquid disclosed herein, such as an aqueous solution.

A crosslinked graft copolymer as comprised in a composition herein canhave a WRV of about, or at least about, 400 in some embodiments. Forinstance, WRV herein can be about, or at least about, 400, 500, 600,700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, or3300.

Absorption of an aqueous liquid by a crosslinked graft copolymer ascomprised in a composition herein can be optionally gauged by measuringcentrifugal retention capacity (CRC) as disclosed in Example 8 below orin U.S. Pat.. No. 8859758 (incorporated herein by reference), forexample. A CRC value herein can be provided in terms of grams of aqueousfluid per grams of crosslinked graft copolymer (“g/g”). A crosslinkedgraft copolymer can have a CRC of about, or at least about, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 28-33, 28-32, 20-25, 21-24, or 22-24 g/g in someembodiments. A corresponding WRV can be made by multiplying a CRCmeasurement by 100, if desired. Further yet, absorption herein canoptionally be measured by determining absorption under load (AUL), suchas via the methodology disclosed in U.S. Pat.. No. 8859758 or EDANA(European Disposables and Non-woven Association) standard test WSP242.2.R3 (12), which are both incorporated herein by reference. AULmeasurements can be provided in terms of grams of aqueous fluid pergrams of crosslinked graft copolymer (“g/g”), and can be measured undera suitable pressure (e.g., psi of about 0.5-1.0, 0.75-1.0, 0.80-0.85, or0.82).

The absorbency of a crosslinked graft copolymer is contemplated in mostor all aspects to be greater than the absorbency of the graft copolymeras it existed before being crosslinking to form the crosslinked graftcopolymer. For example, the absorbency of a crosslinked graft copolymercan be at least about 2, 3, 4, 5, 6, 7, or 8 times greater than theabsorbency of the graft copolymer as it existed before beingcrosslinking.

Absorption herein can optionally be characterized in terms of themaximum amount of aqueous liquid that can be soaked into and retained bya certain amount of crosslinked graft copolymer. A crosslinked graftcopolymer with an absorption capacity of at least 15, 20 or 15-20 g(gram) aqueous liquid/g crosslinked graft copolymer can be characterizedas being superabsorbent in some aspects.

A composition comprising a crosslinked graft copolymer as presentlydisclosed can be in the form of, or comprised within, a personal careproduct, household product, medical product, or industrial product, forexample. In this context, compositions in certain embodiments can beused as absorbent or superabsorbent materials, depending on the degreeof absorption exhibited by the constituent crosslinked graft copolymer.A personal care product, household product, medical product, orindustrial product herein is optionally designed, at least in part, forhandling aqueous liquid absorption.

Examples of personal care products and/or uses herein include absorbentpersonal hygiene products such as baby diapers, potty trainingpants/liners, incontinence products (e.g., pads, adult diapers), andfeminine hygiene products (e.g., sanitary napkins/pads, tampons,interlabial products, panty liners). Thus, a personal care product insome aspects can be characterized as a personal care absorbent articlethat can be placed against or near the skin to absorb and contain afluid discharged or emitted from the body. Examples of personal careproducts that can be adapted accordingly to take advantage of theabsorbency of a crosslinked graft copolymer herein (e.g., replace orsupplement originally used absorbent material in a product) aredisclosed in WO1999/037261; U.S. Pat. Appl. Publ. Nos. 2004/0167491,2009/0204091, 2001/0014797, 2013/0281949, 2002/0087138, 2010/0241098,2011/0137277 and 2007/0287971; and U.S. Pat. Nos. 4623339, 2627858,3585998, 3964486, 6579273, 6183456, 5820619, 4846824, 4397644, 4079739,8987543, 4781713, 5462539, 8912383, 3749094, 3322123, 4762521 and5342343, all of which patent application and patent publications areincorporated herein by reference.

Examples of industrial products and/or uses herein include cablewrappings (e.g., wrappings for power or telecommunication cables); foodpads; agricultural and forestry applications such as for retaining waterin soil and/or to release water to plant roots; firefighting devices;and cleanup of acidic or basic aqueous solutions spills. Examples ofindustrial products that can be adapted accordingly to take advantage ofthe absorbency of a crosslinked graft copolymer herein are disclosed inU.S. Pat. Appl. Publ. Nos. 2002/0147483, 2006/0172048, 20050008737,2008/0199577, 2012/0328723 and 2004/0074271; and U.S. Pat. Nos. 5906952,7567739, 5176930, 6695138, 4865855, 7459501, 5456733, 9089730, 5849210,7670513, 7670513, 5683813, 5342543, 4840734 and 4894179, all of whichpatent application and patent publications are incorporated herein byreference.

Examples of medical products and/or uses herein include wound healingdressings such as bandages and surgical pads; hospital bed sheets;sanitary towels; controlled drug release devices; cell immobilizationislets; three-dimensional cell culture substrates; bioactive scaffoldsfor regenerative medicine; stomach bulking devices; and disposal ofcontrolled drugs. Examples of medical products that can be adaptedaccordingly to take advantage of the absorbency of a crosslinked graftcopolymer herein are disclosed in WO1998/046159; U.S. Pat. Appl. Publ.Nos. 2005/0256486, 20030070232 and 20040128764; and U.S. Pat. Nos.6191341, 7732657, 4925453, 9161860, 3187747 and 5701617, all of whichpatent application and patent publications are incorporated herein byreference.

Personal care products, household products, and/or medical products insome embodiments herein can absorb a bodily fluid such as urine, blood,blood serum, liquid fecal matter (e.g., diarrhea), bile, stomachacid/juice, vomit, amniotic fluid, breast milk, cerebrospinal fluid,exudate, lymph, mucus (e.g., nasal drainage, phlegm), peritoneal fluid,pleural fluid, pus, rheum, saliva, sputum, synovial fluid, sweat, and/ortears.

A composition as presently disclosed can comprise about, or at leastabout, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 99, 99.5, or 99.9 wt%, for example,of one or more crosslinked graft copolymers herein. Dry compositions incertain aspects can be in the form of powder, granules, microcapsules,flakes, or any other form of particulate matter. Other examples includelarger compositions such as pellets, bars, kernels, beads, tablets,sticks, or other agglomerates. A dry composition herein typically hasless than 3, 2, 1, 0.5, or 0.1 wt% water comprised therein.

An absorption method is presently disclosed that comprises, at least,contacting a crosslinked graft copolymer herein with an aqueousliquid-comprising composition, wherein the composition absorbs aqueousliquid from the liquid-comprising composition. An aqueousliquid-comprising composition can be any as disclosed herein. Forexample, such a composition can be urine, blood, blood serum, liquidfecal matter, bile, stomach acid/juice, vomit, amniotic fluid, breastmilk, cerebrospinal fluid, exudate, lymph, mucus, peritoneal fluid,pleural fluid, pus, rheum, saliva, sputum, synovial fluid, sweat, tears,water, or saline.

In certain alternative embodiments, a composition can comprise a verylarge dextran (very high molecular weight dextran) that has beencrosslinked. It is believed that most or all of the conditions disclosedherein for crosslinking a graft copolymer can be applied to crosslinkingany very large dextran (one that is not already comprised in a graftcopolymer herein) as disclosed above, in the below Examples, and in U.S.Pat. Appl. Publ. No. 2016/0122445, which is incorporated herein byreference. It is also believed that a crosslinked very large dextran canbe used in any aqueous liquid absorption application (e.g.,superabsorbent) or method disclosed herein. Accordingly, any of thefeatures of the present disclosure regarding crosslinking graftcopolymers can likewise characterize embodiments in which a very largedextran is crosslinked and utilized, insofar as would be consideredsuitable by a skilled artisan. For example, insofar as would beconsidered suitable by a skilled artisan, the term “graft copolymer” asused in the present disclosure can optionally be replaced with the term“very large dextran” or “very high molecular weight dextran”. Whilecompositions with a crosslinked very large dextran herein are typicallyindependent from those comprising a crosslinked graft copolymer, someembodiments herein are drawn to compositions comprising both types ofcrosslinked material (i.e., crosslinked very large dextran andcrosslinked graft copolymer).

Non-limiting examples of compositions and methods disclosed hereininclude:

1. A composition comprising a crosslinked graft copolymer, wherein thegraft copolymer portion of the crosslinked graft copolymer comprises:(i) a backbone comprising dextran, and (ii) poly alpha-1,3-glucan sidechains comprising at least about 50% alpha-1,3-glucosidic linkages.

2. The composition of embodiment 1, wherein one or more crosslinks ofthe crosslinked graft copolymer are covalent.

3. The composition of embodiment 1 or 2, wherein one or more crosslinksof the crosslinked graft copolymer comprise phosphorus.

4. The composition of embodiment 3, wherein one or more crosslinks ofthe crosslinked graft copolymer comprise a phosphodiester bond.

5. The composition of embodiment 1, 2, 3, or 4, wherein the graftcopolymer portion of the crosslinked graft copolymer comprises at leastabout 50 wt% dextran.

6. The composition of embodiment 1, 2, 3, 4, or 5, wherein the dextranhas a weight-average molecular weight (Mw) of at least about 100000Daltons.

7. The composition of embodiment 1, 2, 3, 4, 5, or 6, wherein the polyalpha-1,3-glucan side chains comprise at least about 95%alpha-1,3-glucosidic linkages.

8. The composition of embodiment 1, 2, 3, 4, 5, 6, or 7, wherein the Mwof one or more individual poly alpha-1,3-glucan side chains is at leastabout 100000 Daltons.

9. The composition of embodiment 1, 2, 3, 4, 5, 6, 7, or 8, wherein thedextran comprises: (i) about 87-93 wt% glucose linked at positions 1 and6; (ii) about 0.1-1.2 wt% glucose linked at positions 1 and 3; (iii)about 0.1-0.7 wt% glucose linked at positions 1 and 4; (iv) about7.7-8.6 wt% glucose linked at positions 1, 3 and 6; and (v) about0.4-1.7 wt% glucose linked at: (a) positions 1, 2 and 6, or (b)positions 1, 4 and 6; wherein the Mw of the dextran is about 50-200million Daltons.

10. The composition of embodiment 9, wherein the Mw of the dextran is atleast about 100 million Daltons.

11. The composition of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,wherein the crosslinked graft copolymer has a centrifugal retentioncapacity (CRC) of at least about 6 gram (g) aqueous fluid per gram (g)crosslinked graft copolymer.

12. The composition of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11,wherein the crosslinked graft copolymer is produced by contacting thegraft copolymer portion with a crosslinking agent and a solvent,optionally wherein the crosslinking agent comprises phosphoryl chloride,and/or the solvent is an aqueous solvent.

13. The composition of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or12, wherein the composition is a personal care product, household careproduct, medical product, or industrial product.

14. A method of producing a crosslinked graft copolymer (such as in anyone of embodiments 1-13), the method comprising: (a) contacting at leasta solvent, a crosslinking agent, and a graft copolymer, wherein thegraft copolymer comprises: (i) a backbone comprising dextran, and (ii)poly alpha-1,3-glucan side chains comprising at least about 50%alpha-1,3-glucosidic linkages, whereby a crosslinked graft copolymer isproduced; and (b) optionally, isolating the crosslinked graft copolymerproduced in step (a).

15. The method of embodiment 14, wherein the solvent is aqueous.

16. A composition comprising crosslinked dextran, wherein the dextrancomprises: (i) about 87-93 wt% glucose linked at positions 1 and 6; (ii)about 0.1-1.2 wt% glucose linked at positions 1 and 3; (iii) about0.1-0.7 wt% glucose linked at positions 1 and 4; (iv) about 7.7-8.6 wt%glucose linked at positions 1, 3 and 6; and (v) about 0.4-1.7 wt%glucose linked at: (a) positions 1, 2 and 6, or (b) positions 1, 4 and6; wherein the weight-average molecular weight (Mw) of the dextran isabout 50-200 million Daltons.

17. The composition of embodiment 16, wherein one or more crosslinks ofthe crosslinked dextran are covalent.

18. The composition of embodiment 16 or 17, wherein one or morecrosslinks of the crosslinked dextran comprise phosphorus.

19. The composition of embodiment 18, wherein one or more crosslinks ofthe crosslinked dextran comprise a phosphodiester bond.

20. The composition of embodiment 16, 17, 18, or 19, wherein the dextranhas an Mw of at least about 100 million Daltons.

21. The composition of embodiment 16, 17, 18, 19, or 20, wherein thecrosslinked dextran has a centrifugal retention capacity (CRC) of atleast about 6 g aqueous fluid per g crosslinked graft copolymer.

22. The composition of embodiment 16, 17, 18, 19, 20, or 21, wherein thecrosslinked dextran is produced by contacting the dextran with acrosslinking agent and a solvent, optionally wherein the crosslinkingagent comprises phosphoryl chloride, and/or the solvent is an aqueoussolvent.

23. The composition of embodiment 16, 17, 18, 19, 20, 21, or 22, whereinthe composition is a personal care product, household care product,medical product, or industrial product.

24. A method of producing a crosslinked dextran (such as in any one ofembodiments 16-23), the method comprising: (a) contacting at least asolvent, a crosslinking agent, and the dextran of embodiment 16, wherebya crosslinked dextran is produced; and (b) optionally, isolating thecrosslinked dextran produced in step (a). 25. The method of embodiment24, wherein the solvent is aqueous.

EXAMPLES

The present disclosure is further exemplified in the following Examples.It should be understood that these Examples, while indicating certainpreferred aspects herein, are given by way of illustration only. Fromthe above discussion and these Examples, one skilled in the art canascertain the essential characteristics of the disclosed embodiments,and without departing from the spirit and scope thereof, can makevarious changes and modifications to adapt the disclosed embodiments tovarious uses and conditions.

Example 1 Poly Alpha-1,3-Glucan Synthesis from High Molecular WeightDextran Primer

This Example describes synthesis of poly alpha-1,3-glucan with aglucosyltransferase enzyme using commercially available dextran withhigh weight-average molecular weight (average 150 kDa) as a primer.Graft copolymers comprising a dextran backbone and poly alpha-1,3-glucanside chains were produced.

Two separate poly alpha-1,3-glucan polymerizations were performed withreactions (A and B) comprising water, sucrose (~100 g/L), dextran, and aStreptococcus salivarius-based glucosyltransferase enzyme thatsynthesizes poly alpha-1,3-glucan with all or nearly allalpha-1,3-glucosidic linkages. Examples of glucosyltransferases that canbe used in such reactions include those disclosed in U.S. Pat. Appl.Publ. No. 2014/0087431, which is incorporated herein by reference (e.g.,SEQ ID NO:4 or 8 therein).

Each of reactions A and B was prepared by mixing 940 g (gram) DI(deionized) water, 100 g sucrose (OmniPur Calbiochem 8550; Lot VF20C; FW342.30), and 1.36 g potassium monophosphate (MW 136.09; Sigma P5379).The pH was measured to be 5.6 using a conductivity meter, and adjusteddown to 5.54 using a few drops of 1 N H₂SO₄. A 1-mL sample was taken forHPLC time point zero (pre-addition of dextran). Then, 5 g and 10 g of150-kDa (avg) dextran (Sigma D4876) were added to reactions A and B,respectively. 500-mL of each reaction was loaded into individual flasks.

After mixing each reaction at about 190 RPM to dissolve the addeddextran, 1-mL HPLC samples were taken from each reaction for time pointzero (post-addition of dextran) analysis. Each reaction was placed intoa circulating heater/chiller set to 25° C. and stirring was commenced at150 rpm. The reactions were allowed to come up to temperature (~24.4°C.) and stirred for about 45 min before enzyme addition. 50 U ofglucosyltransferase enzyme was then added to each reaction.

Filtrate samples (i.e., liquid separated from insoluble products) (1 mL)from each of reactions A and B were taken for HPLC at 2 hr and at theend of each reaction (24 hr). The samples were deactivated for HPLC byheat quenching at 90° C. for 10 min. The samples were filtered through0.45-µm PTFE filters and diluted for HPLC analysis.

Two identical dilutions were made for all of the time-point filtratesamples, with the exception of the 2-hr and 24-hr samples of reaction B.Samples A 2-hr, B 2-hr, and B 24-hr were all very difficult to filterthrough the 0.45-µm PTFE filters. All the samples were run in duplicatein various HPLC columns.

A whole-reaction sample (50-ml) was taken at 2 hr from each of reactionsA and B and suction-filtered as dry as possible through aplastic-disposable filter. Before washing the insoluble products twicewith 50 mL of hot water, the filtrate was removed and saved separately.The insoluble polymer samples were saved in glass vials and stored at10° C. before analyzing by size-exclusion chromatography (SEC) todetermine apparent DP (degree of polymerization), true DP, apparent IV(inherent viscosity), and true IV. Excess insoluble polymer from each2-hr sample was dried in a vacuum oven at 60° C. under nitrogen for 3days, and weighed to determine percent solids.

Pulling the filtrate from the synthesized polymer with suction tooklonger than expected, and was likely related to the continued productionof insoluble polymer in the filtrate, which still contained sucrose andglucosyltransferase enzyme. Once the filtrate was all collected, a 1-mLsample was taken and deactivated for HPLC (see above), while theremainder was deactivated in a 70-80° C. water bath for 15 minutes,allowed to cool, and then filtered to remove insoluble polymer products.

The filtrate was then placed in dialysis tubing (14 kDa molecular weightcut-off [MWCO]) and dialyzed for 2 days in running water to removemonosaccharides (fructose, glucose) and oligomers (DP 2-7). Some minorsolids were formed during dialysis, so the contents were first filteredand then rotary-evaporated (rotovapped) to a liquid concentrate, whichwas frozen in liquid nitrogen. The frozen concentrate was thenlyophilized for 2-3 days, after which the dry solids were weighed andanalyzed by SEC.

After 24 hr, the polymer product slurries created in each of reactions Aand B were suction-filtered. Each filtrate was saved separately and anHPLC sample was taken. The polymer was washed twice with 500 mLdistilled water (room temperature), after which gross water was suckedoff leaving a wet cake. The wet cake was weighed and a sample thereofwas taken for SEC analysis. A wet cake sample (~5-6 g) was oven-dried(60° C. for 3 days) and the total insoluble polymer product yield wascalculated based on initial wet cake weight. The remaining polymer wetcake was frozen for later analysis. The remaining filtrate wasdeactivated in a 90° C. water bath for 15 min and dialyzed for 2 days inrunning water as above. The dialysate was filtered, rotovapped to ~80mL, lyophilized, weighed and submitted for SEC. Per HPLC analysis,monosaccharide and oligomer (DP 2-7) generation was normal and similarbetween polymerizations A and B. Wet cake samples were dissolved for SECanalysis by shaking in DMSO/2% LiCl for 10 min at room temperature.

Various aspects of the filtrates and insoluble products of reactions Aand B are provided in Tables 1-4 below.

TABLE 1 Total Solids Present in Filtrate Lyophilized Filtrate Solids*Reaction 2 hr 24 hr A 0.27 g 3.13 q B 0.37 g 4.75 q *includes oligomers

TABLE 2 Sucrose and Dextran Conversion Reaction Sucrose ConversionDextran Conversion A 99.3% 73% B 99.3% 64%

TABLE 3 Dextran Recovered in Filtrate Recovered Dextran Reaction / Timepoint Mn Mw DPw Mz Mw/Mn A / 2 hr 15196 212556 1312 2644592 13.99 A / 24hr 10195 30003 185 72429 2.94 B / 2 hr 15541 190473 1176 1921736 12.26 B/ 24 hr 11340 45326 280 137362 4.00 starting dextran 20120 244127 15071260514 12.13

TABLE 4 Molecular Weight Profile of Dextran-Poly Alpha-1,3-GlucanCopolymer Products Dextran-Poly Alpha-1,3-Glucan Copolymer Reaction /Time point Mn (kDa) Mw (kDa) DPw Mz (kDa) Mw/Mn A / 2 hr 525 1156 71371942 2.2 A / 24 hr 322 1011 6244 2126 3.14 B / 2 hr 465 1005 6202 17652.16 B / 24 hr 285 802 4948 1764 2.81

SEC analysis of the starting dextran used in each reaction showed thatit was branched. It was estimated that there was a pendant glucosebranching from the starting dextran about every 20 monomeric units ofthe dextran. Each polymerization reaction (24 hr) gave a water-insolublepolymer with a high DPw: ~6000 for reaction A (10 g/L dextran loading)and ~5000 for reaction B (20 g/L dextran loading) (Table 4). Polyalpha-1,3-glucan chains grew off of the dextran branch points, forming agraft copolymer (refer to FIGS. 1 and 2 ).

Dextranase degradation analyses indicated that the poly alpha-1,3-glucanside chains each had a DPw of roughly 1000. Briefly, dextranase assayswere conducted by individually reacting dextran-poly alpha-1,3-glucangraft copolymer products with dextranase in a buffered reaction (pH5.3-5.7, room temperature, nutation) for about 4 days.

Thus, considering that the starting dextran had a measured DPw of about1500 (Table 3), and each side chain was about 1000 DPw, there may havebeen on average about 4-5 poly alpha-1,3-glucan chains on each dextran.Based on this observation, it appears that only a small fraction of thependant glucose units of the dextran served to prime polyalpha-1,3-glucan side chain synthesis (i.e., there were likely onlyabout 4-5 side chains, whereas it might have been possible to have hadabout 75 side chains given the presence of a pendant glucose group every20 monomeric units of the dextran [DPw 1507 divided by 20]).

The molecular weight of dextran recovered in filtrate samples of 24-hrreactions was low, in comparison to the starting dextran molecularweight (Table 3). While one hypothesis was that the dextran may havebeen degraded by the glucosyltransferase enzyme in the reaction, thiswas found not to be the case (see Example 3). Thus, it was likely thatthe dextran was effectively fractionated during the reaction, withhigher molecular weight dextran preferentially being used as a substratefor priming poly alpha-1,3-glucan side chain synthesis. Following thisscenario, the larger dextran molecules used to prime synthesis ofinsoluble graft copolymer would have been removed from the soluble pool,leaving behind smaller dextran molecules in reaction filtrates. Thisobservation is intriguing, especially given that other work(WO15/119859) suggested that dextran molecular weight does not play arole in dextran priming of 1,3-glucosidic link-comprising glucansynthesis by glucosyltransferase enzymes.

Thus, graft copolymers comprising a dextran backbone and polyalpha-1,3-glucan side chains were produced. Each of these graftcopolymers can optionally be crosslinked following the proceduresdisclosed in Example 8 below, for example. It is potentially of interestthat there were relatively few side chains (4-5), considering that,theoretically, there could have been at least 10-15 times more sidechains synthesized. Also, in reactions for preparing this graftcopolymer, it appears that high molecular weight dextran, as opposed tolower molecular weight dextran, is preferentially used as a substrate byglucosyltransferases that synthesize glucan comprising mostlyalpha-1,3-glucosidic linkages.

Example 2 Controlling the Molecular Weight and Polydispersity ofDextran-Poly Alpha-1,3-Glucan Graft Copolymer Products of aGlucosyltransferase Enzyme Reaction

This Example is in addition to Example 1, which together demonstrate,for example, that the molecular weight and polydispersity ofdextran-poly alpha-1,3-glucan graft copolymer product can be controlledby modifying the concentration of dextran entered into aglucosyltransferase enzyme reaction.

In general, except as noted below, the procedures described in Example 1were applied to synthesize and analyzed dextran-poly alpha-1,3-glucancopolymers.

Briefly, two 500-mL glucan synthesis reactions were run at about 25° C.with 100 g/L sucrose and 100 U/L of an S. salivarius-basedglucosyltransferase enzyme that synthesizes poly alpha-1,3-glucan withall or nearly all alpha-1,3-glucosidic linkages with stirring at 150rpm. To set up these reactions, 100 g of sucrose (OmniPur Calbiochem8550) and 1.36 g of potassium monophosphate (Sigma P5379) were dissolvedin 940 g tap water and adjusted to pH 5.5 with NaOH. A 1-mL sample (t=0)was taken for HPLC analysis after which the solution was divided in two500-mL portions. Flasks for reactions A and B were each charged with 500mL of the sucrose solution and 1.25 g or 2.5 g, respectively, of 150-kDa(avg) dextran (Sigma D4876). HPLC (t=0) samples were taken after whichthe glucosyltransferase enzyme was added.

At 2 hr post enzyme addition, 50-mL samples (reaction solution andinsoluble product) were taken from each of reactions A and B andsuction-filtered. The filtrates were saved; 1 mL of each filtrate wasremoved for HPLC (t=2 hr). The insoluble polymer products were washedtwice with 50 mL hot water and analyzed by SEC. The filtrates weredeactivated in an 80° C. water bath for 15 min, refiltered and dialyzed(14 kDa MWCO) for 18 days in running water to remove monosaccharides(fructose, glucose) and oligomers (DP 2-7).

At 24 hr post enzyme addition, the polymer product slurries created ineach of reactions A and B were heated to 65° C. in a circulating bathand stirred for 1 hr to deactivate the enzyme. The slurries were thensuction-filtered; each filtrate was saved and a 1-mL sample (t=24 hr)was taken for HPLC analysis. The polymer was washed, after which grosswater was sucked off leaving a wet cake. The wet cake was weighed and asample thereof was taken for SEC analysis. A wet cake sample wasoven-dried (60° C. for 3 days) and the total insoluble polymer productyield was calculated based on initial wet cake weight. The remainingpolymer wet cake was frozen for later analysis. The filtrate wasdialyzed for 17 days in running water as above. The dialysate wasfiltered, rotovapped to ~80 mL, lyophilized, weighed and submitted forSEC. Per HPLC analysis, monosaccharide and oligomer (DP 2-7) generationwas normal and similar between polymerizations A and B.

Various aspects of the filtrates and insoluble products of reactions Aand B of this Example are provided in Tables 5-7 below.

TABLE 5 Total Solids Present in Filtrate Lyophilized Filtrate Solids*Reaction 2 hr 24 hr A 0.057 g 0.086 g B 0.099 g 0.322 g *monosaccharidesand oligomers removed

TABLE 6 Sucrose and Dextran Conversion Reaction Sucrose Conversion MassBalance Dextran Conversion A 99.3% 98.2% 92% B 99.4% 99.0% 86%

TABLE 7 Molecular Weight Profile of Dextran-Poly Alpha-1,3-GlucanCopolymer Products Dextran-Poly Alpha-1,3-Glucan Copolymer Reaction Timepoint Starting Dextran Concentration Mn (kDa) Mw (kDa) DPw Mz (kDa)Mw/Mn A (Example 2) 24 hr 2.5 g/L 261 1198 7394 2520 4.59 B (Example 2)24 hr 5 g/L 301 1236 7629 2518 4.10 A (Example 1) 24 hr 10 g/L 322 10116244 2126 3.14 B (Example 1) 24 hr 20 g/L 285 802 4948 1764 2.81A(Example 2) 2 hr 2.5 g/L 177 1762 10873 2814 9.96 B(Example 2) 2 hr 5g/L 259 1499 9255 2555 5.78 A (Example 1) 2 hr 10 g/L 525 1156 7137 19422.2 B (Example 1) 2 hr 20 g/L 465 1005 6202 1765 2.16

Each polymerization reaction after 24 hr in this Example producedwater-insoluble polymer with a high DPw of about 7500 (Table 7). Thepolydispersities (Mw/Mn) of the insoluble polymer products wererelatively high, especially for reactions with less starting dextran(Table 7), suggesting there is poly alpha-1,3-glucan homopolymer presentin the insoluble products in addition to dextran-poly alpha-1,3-glucangraft copolymer. Such a result was to be expected in a system starvedfor dextran; indeed, reactions with higher amounts of starting dextran(Example 1) yielded products with lower polydispersity (Table 7). Itthus appears that the polydispersity of a dextran-poly alpha-1,3-glucangraft copolymer produced herein can be controlled as a function of thelevel of dextran entered into a glucosyltransferase reaction.

FIG. 3 shows, for 24-hr reactions in which most of the starting dextranhas been consumed, the relationship between starting dextranconcentration and DPw of the dextran-poly alpha-1,3-glucan graftcopolymer product formed. Homopolymerization of poly alpha-1,3-glucanalone competes with dextran priming at low dextran concentrations, whileeach of the dextran chains gets fewer glucan grafts at higher dextranconcentrations. The maximum graft copolymer molecular weight, appears tobe produced when using 5 g/L dextran (FIG. 3 , Table 7) in a reactionhaving 100 g/L sucrose and 100 U/L glucosyltransferase enzyme. It thusappears that the molecular weight of a dextran-poly alpha-1,3-glucangraft copolymer produced herein can be controlled as a function of thelevel of dextran entered into a glucosyltransferase reaction.

Thus, graft copolymers comprising a dextran backbone and polyalpha-1,3-glucan side chains were produced. Each of these graftcopolymers can optionally be crosslinked following the proceduresdisclosed in Example 8 below, for example. Also, the molecular weightand polydispersity of dextran-poly alpha-1,3-glucan copolymer productscan be controlled by modifying the concentration of dextran entered intoa glucosyltransferase enzyme reaction.

Example 3 Glucosyltransferase Enzyme Activity Does Not Degrade Dextran

This Example demonstrates that the glucosyltransferase used in Examples1 and 2 to synthesize dextran-poly alpha-1,3-glucan graft copolymer doesnot degrade dextran. Therefore, the apparent dextran partitioning effectobserved in the above reactions was not due to dextran degradation fromglucosyltransferase activity.

As described in Example 1, when poly alpha-1,3-glucan synthesis with aglucosyltransferase enzyme is primed with dextran, the recoveredunreacted dextran has a significantly lower molecular weight than thedextran which was initially used in the reaction. It was not knownwhether the dextran was effectively fractionated in theglucosyltransferase reaction - preferentially reacting larger dextranchains to form insoluble dextran-poly alpha-1,3-glucan copolymer,leaving smaller unreacted dextran chains in the reaction solution - orwhether the glucosyltransferase enzyme was capable of degrading thedextran.

The purpose of this experiment was to examine if exposing dextran to theglucosyltransferase enzyme used in Examples 1 and 2 under normalreaction conditions, but without sucrose, would lead to dextrandegradation. 2.5 g of 150-kDa (avg) dextran (Sigma D4876) and 0.68 g ofpotassium monophosphate (Sigma P5379) were dissolved in 490 g tap waterto provide a solution at pH 5.59. This solution was stirred at 25° C. ina reactor after which 50 U of the glucosyltransferase enzyme was added.The solution was then stirred at 150 rpm for 24 hr and then rotovappedfrom a hot water bath to leave a damp solid. The solid was taken up in20 mL of distilled water and the resulting hazy solution was clarifiedby suction-filtration; a very small amount (~0.1 g) of light brownsolids was removed. The filtrate was lyophilized to recover 2.87 gdextran, which was analyzed by SEC and compared with the startingdextran (Table 8).

TABLE 8 Analysis of Dextran Molecular Weight Before and After Exposureto Glucosyltransferase Enzyme Dextran Mn (kDa) Mp (kDa) Mw (kDa) Mz(kDa) Mw/Mn DPw Starting 60.07 83.6 258 1221 4.29 1593 Recovered 58.0183.6 249 1255 4.30 1537

The results in Table 8 show that the glucosyltransferase enzyme does notdegrade dextran under the reaction conditions employed in Examples 1 and2 (but without sucrose). This result indicates that the enzymaticprocess of poly alpha-1,3-glucan grafting onto dextran effectively actsto fractionate the dextran based on molecular weight as described above.

Example 4 Poly Alpha-1,3-Glucan Synthesis from Lower Molecular WeightDextran

This Example describes synthesis of poly alpha-1,3-glucan with aglucosyltransferase enzyme using commercially available dextran primerwith a weight-average molecular weight of about 40 kDa.

The purpose of this experiment was to synthesize a dextran-polyalpha-1,3-glucan graft copolymer using dextran having a lower molecularweight than the dextran used in Examples 1 and 2. The dextran used inthis experiment has a molecular weight of about 35-45 kDa, which isroughly four times less than the molecular weight of the dextranemployed in Examples 1 and 2.

A 1000-mL poly alpha-1,3-glucan polymerization reaction was performed asfollows. Sucrose (100 g; OmniPur Calbiochem 8550), dextran (10 g, 35-45kDa, DPw = 220-280, Sigma D1662) and potassium monophosphate (1.36 g,Sigma P5379) were dissolved in 940 g of tap water to give pH 5.67.Stirring at 25° C./150 rpm was then commenced after which 100 U of theglucosyltransferase used in the above Examples was added; stirring at25° C./150 rpm was continued for 24 hr. After 1.5 hr, a 50-mL insolubleproduct sample was suction-filtered, washed and suctioned to a damp wetcake (8.7 g) and submitted for SEC analysis. At 24 hr, the insolubleproduct slurry was suction-filtered and washed three times with 500 mLhot tap water. The gross water was suction-removed and the wet cake wasweighed (480 g). Wet cake samples were taken for SEC analysis andpercent solids determination (7.6 wt%), the latter of which was done byoven-drying (60° C. for 3 days). The total insoluble dextran-polyalpha-1,3-glucan product yield was calculated based on initial wet cakeweight and percent solids. The molecular weight profile of eachinsoluble product at 1.5 hr and 12 hr was determined (Table 9).

TABLE 9 Molecular Weight Profile of Dextran-Poly Alpha-1,3-GlucanCopolymer Products Dextran-Polv Alpha-1,3-Glucan Copolymer Time point Mn(kDa) Mp (kDa) Mw (kDa) DPw Mz (kDa) Mw/Mn 1.5 hr 359 478 593 3660 9461.65 24 hr 210 365 474 2926 836 2.26

Based on measured DPw, it appears that two or at most three polyalpha-1,3-glucan side chains were synthesized on the dextran. Thisresult seems interesting, since dextran (150-kDa avg, Example 1) roughlyfour times larger than the dextran (40 kDa) used in this Example hadabout 4-5 poly alpha-1,3-glucan side chains synthesized thereupon (seeExample 1). If 2-3 side chains could be synthesized on a 40 kDa dextran,it might have been expected that about 8-12 side chains (instead of 4-5)would have been synthesized on a 150-kDa dextran.

As shown in this Example, dextran of about 40 kDa could be used to primepoly alpha-1,3-glucan side chain synthesis. This result is noteworthy inview of Example 1, which shows that dextran of similar molecular weightdid not prime such side chain synthesis when in the presence of largerdextran molecules. The partitioning effect observed in Example 1 (largerdextran preferentially used to prime synthesis of insoluble product,whereas smaller dextran remained in solution) is thus furtherintriguing, given the results of the present Example showing thatsmaller molecular weight dextran, when alone, can prime polyalpha-1,3-glucan side chain synthesis. Regardless of these insights,each of the graft copolymers produced in this Example can optionally becrosslinked following the procedures disclosed in Example 8 below, forexample.

Example 5 Poly Alpha-1,3-Glucan Polymerization from Very High MolecularWeight Dextran Primer

This Example describes synthesis of poly alpha-1,3-glucan with aglucosyltransferase enzyme using dextran with very high weight-averagemolecular weight (at least 50 million Daltons). Graft copolymercomprising a very large dextran backbone and poly alpha-1,3-glucan sidechains was produced. Dextran with Very High Molecular Weight

Dextran with a very high weight-average molecular weight was firstprepared as described in U.S. Appl. Publ. No. 2016/0122445 (Example 9therein, which employed GTF 0768), which is incorporated herein byreference, but using 300 g/L sucrose (instead of 100 g/L sucrose). Thelinkage structure of this dextran was believed to be consistent with thelinkage structure disclosed in US2016/0122445 (Example 9 therein); Table10 lists the linkages for samples initially dissolved in DMSO or DMSO/5%LiCl as disclosed in US2016/0122445 (Example 9 therein).

TABLE 10 Linkage Profile of Very High Molecular Weight Dextran Wt%/Mol%of Glucose Monomers in Dextran Sample 3-glc^(a) 6-glc^(b) 4-glc^(c)3,6-glc^(d) 2,6- + 4,6-glc^(e) DMSO 0.4 90.2 0.4 8.3 0.7 DMSO/5% LiCl0.9 89.3 0.4 8.0 1.4 ^(a) Glucose monomer linked at carbon positions 1and 3. ^(b) Glucose monomer linked at carbon positions 1 and 6. ^(c)Glucose monomer linked at carbon positions 1 and 4. ^(d) Glucose monomerlinked at carbon positions 1, 3 and 6. ^(e) Glucose monomer linked atcarbon positions 1, 2 and 6, or 1, 4 and 6.

Based on this information and some other studies (data not shown), it iscontemplated that this product is a branched structure in which thereare long chains (containing mostly or all alpha-1,6-linkages) of about20 DP in length (average) that iteratively branch from each other (e.g.,a long chain can be a branch from another long chain, which in turn canitself be a branch from another long chain, and so on). The branchedstructure also appears to comprise short branches from the long chains;these short chains are believed to be 1-3 DP in length and mostlycomprise alpha-1,3 and -1,4 linkages, for example. Branch points in thedextran, whether from a long chain branching from another long chain, ora short chain branching from a long chain, appear to comprise alpha-1,3,-1,4, or -1,2 linkages off of a glucose involved in alpha-1,6 linkage.Roughly 25% of all the branch points of the dextran branched into a longchain.

The molecular weight and other size features of the dextran prepared inthe present Example were believed to be consistent with the featuresdisclosed in US2016/0122445 (Example 9 therein), which were as follows:a weight-average molecular weight (Mw) of 1.022 (+/- 0.025) × 10⁸ g/mol(i.e., roughly 100 million Daltons) (from MALS analysis), a z-averageradius of gyration of 243.33 (+/-0.42) nm (from MALS analysis), az-average hydrodynamic radius of 215 nm (from QELS analysis), and astandard deviation of particle size distribution (PSD) of about 0.259(from QELS analysis, indicating polydispersity in terms of hydrodynamicsize). The very high molecular weight dextran produced in this Examplecan optionally be used in the procedures described below in Example 9for preparing crosslinked dextran.

Dextran-Poly Alpha-1,3-Glucan Graft Copolymers Comprising Very HighMolecular Weight Dextran

The very high weight-average molecular weight dextran prepared above wasused in the following enzymatic reaction to prepare dextran-polyalpha-1,3-glucan graft copolymer.

A 500-mL poly alpha-1,3-glucan synthesis reaction was run at 25° C. withstirring at 150 rpm using 100 g/L sucrose, 9.8 g/L dextran and 100 U/Lof the glucosyltransferase used in the above Examples. To set up thisreaction, dextran (4.9 g) was ground in a mortar and pestle and stirredat 50° C. with 470 g of tap water for 16 hr to give a hazy solution.Then sucrose (50 g, OmniPur Calbiochem 8550) and potassium monophosphate(0.68 g, Sigma P5379) were added and dissolved with stirring to give pH5.75. The solution was stirred at 25° C. in a reactor, after which theglucosyltransferase enzyme (50 U) was added. In about half an hour, thereaction had become a suspension of firm, spongy particles of about 5 mmin size.

At 2 hr, a 50-mL sample was removed from the reaction (the polymerparticles clogged the pipette, so not much insoluble product wasobtained). This sample (a suspension) stood for a couple of hours beforeit was suction-filtered, washed and suctioned to a damp wet cake (1.3 g)and submitted for SEC analysis. The sample was not deactivated to killenzyme activity, so additional poly alpha-1,3-glucan likely formedbefore it was suction-filtered. The filtrate was heated to deactivatethe enzyme in it. The reaction was continued to 24 hr, after which theinsoluble product slurry was suction-filtered; the filtrate (350 mL) wassaved and analyzed by HPLC.

The initial filtrate was dialyzed by circulating across a MilliporePELLICON 2 PLCTK regenerated cellulose crossflow membrane (30-kDacutoff; 0.1 m²) at 100 mL/min and 10 psig. This dialysis served toremove monosaccharides and oligomers (via permeate), and leaveunreacted, soluble dextran in the retentate. Deionized water wascontinuously added to the recirculating feed to replace water lost topermeate; ultimately, 3500 mL of water was used to wash outmonosaccharides and oligomers. The retentate was then lyophilized torecover <0.1 g unreacted dextran. These results of only a small amountof soluble dextran in the enzyme reaction indicate that most of thedextran was used to prime poly alpha-1,3-glucan synthesis, thus drawingthe dextran to the insoluble products of the reaction.

The insoluble product (dextran-poly alpha-1,3-glucan graft copolymer) ofthe glucosyltransferase reaction was washed three times with 500 mL ofhot tap water; the product consisted of mostly 5-mm particles and asmall amount of fines. The gross water was suction-removed and the dampparticles were weighed (82.8 g; 24-hr sample); product samples wereremoved for SEC and percent solids determination. A wet cake sample(1.105 g) was oven-dried (60° C./2 days) for this purpose. The isolateddextran-poly alpha-1,3-glucan graft copolymer comprised about 25%dextran and 75% poly alpha-1,3-glucan.

Dextranase degradation analyses (performed as described in Example 1)indicated that the poly alpha-1,3-glucan side chains of the synthesizedcopolymer each had a DPw of roughly 1000. This side chain lengthmolecular weight estimate is the same as that observed for side chainssynthesized from lower molecular weight dextran (Example 1-2).

Thus, graft copolymer comprising (i) a very large, branched dextranbackbone and (ii) poly alpha-1,3-glucan side chains was produced. Suchgraft copolymer can optionally be crosslinked following the proceduresdisclosed in Example 8 below, for example.

Example 6 Preparation of Additional Dextran-Poly Alpha-1,3-Glucan GraftCopolymers Comprising Very High Molecular Weight Dextran

Additional dextran-poly alpha-1,3-glucan graft copolymers comprisingvery high molecular weight dextran were prepared in this Example. Thevery high molecular weight dextran described in Example 5 was used forsynthesizing these additional graft copolymers.

Eight glucosyltransferase (100 U/L) reactions were set up and rungenerally as described in Example 5, but with the followingmodifications. The reactions were run at 25° C. in 1-L reactions stirredat 150 rpm using helical ribbon stirrers. Table 11 lists the amount ofdextran and sucrose entered into each reaction. The pH of the reactionswas 5.2-5.8 and left unadjusted. Insoluble product samples were taken at24 hr after starting the reactions; these samples were worked up byfiltering and washing. The resulting wet cakes were weighed and a samplethereof was dried to determine percent solids and yield. Insolubleproduct samples were analyzed by SEC and NMR. The results of eachreaction are summarized in Table 11.

TABLE 11 Properties of Dextran-Poly Alpha-1,3-Glucan Graft CopolymersProduced in Glucosyltransferase Reactions 1-L Reaction Copolymer ProductProfile Dextran (g) Sucrose (a) Sucrose Converted (%) Copolymer Yield(g) Dextran in Copolymer (wt%) Copolymer Appearance 5 100 100 40.0 10.6Stopped stirring; large particles (~5 mm balls), very coarseparticulate, no fines, settled 5 200 100 68.8 5.9 Large particles (~4mm) suspended in fine slurry; mostly granular 2 100 100 38.4 4.5 Stoppedstirring; large particles remained suspended 5 300 69 78.9 4.1 Smallparticles suspended in fine slurry 3 200 100 67.5 3.9 Particles the sizeof sand grains suspended in fine slurry 3 300 71 84.5 2.4 Largeparticles (~4 mm) suspended in fine slurry 1 200 99 75.8 1.4 Fineparticles; more difficult to filter than above products (rows 1-6) 1 30077 105.3 0.9 Fine particles; more difficult to filter than aboveproducts (rows 1-6)

Overall, graft copolymers with a higher dextran content (e.g., 2.4-10.6wt%) appeared as larger particles and were more easily filtered comparedto their counterparts with lower dextran content (e.g., 0.9-1.4 wt%).FIGS. 4 and 5 show photographs of graft copolymer samples containing10.6 wt% dextran (Table 11, row 1, more filterable) and 0.9 wt% dextran(Table 11, row 8, less filterable), respectively.

Three more glucosyltransferase (100 U/L) reactions were set up and rungenerally as described immediately above, using 100 g/L sucrose anddextran at 2, 5, or 10 g/L. These reactions (24 hr) produceddextran-poly alpha-1,3-glucan graft copolymers comprising, respectively,95%, 87.5%, or 75% alpha-1,3 glucosidic linkages. This product linkageprofile is consistent with the products listed in rows 1 and 3 of Table11, which were made under similar reaction conditions (initial sucroseand dextran levels) (note that the dextran component in each productlargely represents alpha-1,6-glucosidic linkages).

Each of the graft copolymers produced in this Example can optionally becrosslinked following the procedures disclosed in Example 8 below, forexample.

Example 7 Preparation of Additional Dextran-Poly Alpha-1,3-Glucan GraftCopolymers Comprising Very High Molecular Weight Dextran

Yet additional dextran-poly alpha-1,3-glucan graft copolymers comprisingvery high molecular weight dextran were prepared in this Example.

Three separate enzymatic procedures, described below as processes 76, 77and 79, were followed to prepare samples of dextran-polyalpha-1,3-glucan graft copolymer. The weight percentage of very highmolecular weight dextran in each of these graft copolymers was over 50%.

Process 76

To a 22-liter agitated round-bottom flask were added 1978 g of sucrose,4831 g of de-ionized (DI) water, and 5.18 g of potassium phosphatemonobasic. After the solids dissolved, the pH was adjusted to 6.5. Whenthe batch temperature reached 27° C., 4.8 mL of a cell lysate containingGTF 0768 (U.S. Appl. Publ. No. 2016/0122445) was added to commencepolymerization of dextran with very high molecular weight. After 11.8hours, the reaction was heated to 60° C. and held for 30 minutes todeactivate the GTF enzyme. To this preparation were added 9930 g DIwater and 11.8 g of potassium phosphate monobasic. The pH was adjustedto 5.5 and the temperature was stabilized at 33° C. The secondpolymerization (for synthesis of alpha-1,3-glucan side chains from thevery high molecular weight dextran) was then started by adding 42 mL ofa cell lysate comprising an S. salivarius-based glucosyltransferaseenzyme that synthesizes poly alpha-1,3-glucan with all or nearly allalpha-1,3-glucosidic linkages. Immediately after this enzyme addition, afeed of a 56 wt% aqueous sucrose solution was started at a rate of 3.0mL/min. The sucrose solution was fed for 2 hours and 19 minutes. Thereaction was held at 33° C. under agitation for 23.75 hours afterstopping the sucrose addition. The reaction was then heated to 60° C.and held for 30 minutes to deactivate the second enzyme, therebycompleting synthesis of a dextran-poly alpha-1,3-glucan graft copolymerproduct. Gravimetric analysis of the final slurry measured 3.1% product.Proton NMR analysis done in deuterated DMSO containing 2% lithiumchloride indicated that the graft copolymer product contained 61 wt% ofdextran.

Process 77

To a 22-liter agitated round bottom-flask were added 2245 g of sucrose,5721 g of DI water, and 6.05 g of potassium phosphate monobasic. Afterthe solids dissolved, the pH was adjusted to 6.5. When the batchtemperature reached 31° C., 5.6 mL of a cell lysate containing GTF 0768(U.S. Appl. Publ. No. 2016/0122445) was added to commence polymerizationof dextran with very high molecular weight. After 72.9 hours, thereaction was heated to 60° C. and held for 30 minutes to deactivate theGTF enzyme. To this preparation were added 9940 g DI water and 11.8 g ofpotassium phosphate monobasic. The pH was adjusted to 5.5 and thetemperature was stabilized at 33° C. The second polymerization (forsynthesis of alpha-1,3-glucan side chains from the very high molecularweight dextran) was then started by adding 42 mL of a cell lysatecomprising an S. salivarius-based glucosyltransferase enzyme thatsynthesizes poly alpha-1,3-glucan with all or nearly allalpha-1,3-glucosidic linkages. Immediately after this enzyme addition, afeed of a 56 wt% aqueous sucrose solution was started at a rate of 3.0mL/min. The sucrose solution was fed for 3 hours and 28 minutes. Thiswas equivalent to adding 431 g of sucrose. The reaction was then heatedto 60° C. and held for 15 minutes to deactivate the second enzyme,thereby completing synthesis of a dextran-poly alpha-1,3-glucan graftcopolymer product. Proton NMR analysis done in deuterated DMSOcontaining 2% lithium chloride indicated that the graft copolymerproduct contained 82 wt% of dextran.

Process 79

To a 2-liter agitated and jacketed resin vessel were added 295 g ofsucrose, 721 g of DI water, and 0.77 g of potassium phosphate monobasic.After the solids dissolved, the pH was adjusted to 6.5. When the batchtemperature reached 27° C., 0.72 mL of a cell lysate containing GTF 0768(U.S. Appl. Publ. No. 2016/0122445) was added to commence polymerizationof dextran with very high molecular weight. After 71.8 hours, thereaction was heated to 60° C. and held for 15 minutes to deactivate theGTF enzyme. To this preparation were added 1516 g DI water and 1.80 g ofpotassium phosphate monobasic. The pH was adjusted to 5.5 and thetemperature was stabilized at 33° C. The second polymerization (forsynthesis of alpha-1,3-glucan side chains from the very high molecularweight dextran) was then started by adding 6 mL of a cell lysatecomprising an S. salivarius-based glucosyltransferase enzyme thatsynthesizes poly alpha-1,3-glucan with all or nearly allalpha-1,3-glucosidic linkages. Immediately after this enzyme addition, afeed of a 56 wt% aqueous sucrose solution was started at a rate of 41.4mL/hr. A total of 62.5 mL of the aqueous sucrose solution was added.This was equivalent to adding 44.1 g of sucrose. The reaction was heldat 33° C. for 17.6 hours and then heated to 60° C. and held for 15minutes to deactivate the second enzyme, thereby completing synthesis ofa dextran-poly alpha-1,3-glucan graft copolymer product. Proton NMRanalysis done in deuterated DMSO containing 2% lithium chlorideindicated that the graft copolymer product contained 90 wt% of dextran.

Thus, additional samples of dextran-poly alpha-1,3-glucan graftcopolymers were prepared following the above procedures. Each of thesegraft copolymers was separately used in Example 8 below to preparecrosslinked graft copolymers.

Example 8 Preparation of Crosslinked Graft Copolymers for Aqueous LiquidAbsorption Applications

This Example describes crosslinking dextran-poly alpha-1,3-glucan graftcopolymers to yield crosslinked graft copolymers. These crosslinkedgraft copolymers exhibited enhanced absorption of aqueous liquidscompared to their non-crosslinked counterparts.

Crosslinking Reaction

Individual crosslinking reactions were performed following the belowprotocol using dextran-poly alpha-1,3-glucan graft copolymers producedin process 76 (graft copolymer with 61 wt% dextran), process 77 (graftcopolymer with 82 wt% dextran), or process 79 (graft copolymer with 90wt% dextran) of Example 7. For ease of discussion below, these graftcopolymers will be referred to their respective synthesis process number(76, 77, or 79).

1. About 5 g (process-76) or 10 g (process-77 or -79) of graft copolymerwas hydrated in about 45 or 90 g of DI water, respectively, resulting ina mixture comprising about 10 wt% graft copolymer.

2. NaOH solution (50 wt%) was added to the mixture (12 g NaOH for 5 ggraft copolymer, 24 g NaOH for 10 g graft copolymer).

3. The mixture was stirred on a shake table for 30 minutes, during whichtime the graft copolymer dissolved and the viscosity increased. Thecolor of the resulting solution was amber.

4. Two aliquots of freshly distilled phosphoryl chloride (POCl₃) wereslowly added to the solution with vigorous stirring, such that the finalconcentration of POCl₃ was 1.6 wt%, thereby providing a crosslinkingreaction. The crosslinking reaction comprising process-76 graftcopolymer thickened to such an extent that it was necessary to add DIwater to allow stirring to continue during and after POCl₃ addition.

5. The reaction was stirred for 1 hour, during which time it gelled.Then, it was filtered and washed to near neutral conditions (with HCIadjustment as required) until NaCl could not be detected using AgNO₃.

6. The crosslinked graft copolymer product was dried in an 80° C. vacuumoven with a nitrogen flush for about 60 hours. The resulting materialsfor each crosslinking reaction were brittle; each product was ground ina small coffee grinder and stored.

Thus, crosslinked dextran-poly alpha-1,3-glucan graft copolymers wereproduced (referred to below as crosslinked process-76, -77, or -79 graftcopolymer). The product yields for the crosslinked process-76, -77 and-79 graft copolymers were measured to be 95%, 31% and 38%, respectively.

Centrifugal Retention Capacity (CRC) Evaluation

Each crosslinked graft copolymer, as well as its respectivenon-crosslinked counterpart, was tested for aqueous liquid absorptionability via CRC evaluation as follows.

Polysaccharide (crosslinked or non-crosslinked graft copolymer) (200 mg)was heat-sealed into a weighed 50 mm x 80 mm teabag and soaked in DIwater or a 0.9-wt% NaCl solution for 30 minutes. The NaCl solution wasused to simulate urine. The teabag was then placed in a basketcentrifuge and spun at 1878 rpm for 3 minutes. The weights of theteabag, dry polysaccharide, and liquid retained by the teabag weredetermined accordingly. The basket CRC (g liquid retained per g drypolymer) was calculated using the following equation:

$\begin{array}{l}\left\lbrack {\left( \text{weight of teabag and polymer post centrifuge} \right) - \left( {\text{3} \times \text{weight of}} \right)} \right) \\{\left( \left( \text{dry teabag* + weight of dry polymer} \right) \right\rbrack/{\text{weight of dry polymer}\text{.}}}\end{array}$

*Since the teabag picks up additional liquid that could skew theresults, a correction factor of 3-times the teabag weight was determinedexperimentally and applied accordingly in the above formula.

Certain Observations

-   Each of the crosslinked process-76, -77 and -79 graft copolymers    hydrated very well.-   During washing, each of the crosslinked process-76, -77 and -79    graft copolymers swelled a significant amount. This was an    encouraging sign indicating that crosslinking had occurred with    improvements in water absorption.-   CRC evaluation results are reported below in Table 12 (data    represent average of two repeats). In general, each crosslinked    graft copolymer exhibited higher aqueous liquid absorption compared    to its respective non-crosslinked counterpart. It is noted that the    crosslinking reaction for process-76 graft copolymer required a    significant amount of added DI water during POCl₃ addition to enable    adequate stirring. This higher dilution likely accounts for why this    crosslinked graft copolymer exhibited a lower absorption profile    compared to crosslinked process-77 and -79 graft copolymers.

TABLE 12 Absorption of Aqueous Liquid by Dextran-Poly Alpha-1,3-GlucanGraft Copolymers (Crosslinked vs. Non-Crosslinked) Sample CRC (g/g) DIWater 0.9-wt% NaCl Process-76 graft copolymer Non-crosslinked 5.543 5.57Crosslinked 10.914 6.04 Process-77 graft copolymer Non-crosslinked 4.0854 Crosslinked 29.116 9.41 Process-79 graft copolymer Non-crosslinked6.188 6.04 Crosslinked 32.527 10.86

Thus, crosslinked graft copolymers herein exhibit enhanced absorption ofaqueous liquids compared to their non-crosslinked counterparts. Giventhis activity, it is contemplated that crosslinked dextran-polyalpha-1,3-glucan graft copolymers are suitable for use in variousaqueous liquid absorption applications as disclosed above.

Example 9 Preparation of Crosslinked Very High Molecular Weight Dextranfor Aqueous Liquid Absorption Applications

This Example describes crosslinking very high molecular weight dextran(“very large dextran”) using various crosslinking agents.

The dextran samples used in all the crosslinking studies in this Examplewere produced using enzyme GTF 0768 (U.S. Appl. Publ. No. 2016/0122445),as disclosed above in Examples 5 and 7, for example. CRC measurementswere made following the procedure described above in Example 8.Absorption under load (AUL) measurements were made following theprocedure disclosed in EDANA standard test WSP 242.2.R3 (12), which isincorporated herein by reference.

Crosslinking With Phosphoryl Chloride (POCl₃)

Very high molecular weight dextran (2.5 g) was added to water (22.5 g)portion by portion with stirring. The solution was slowly stirred tohomogeneity. Sodium hydroxide solution (6 g, Fisher Scientific, 50%) wasthen added. The resulting preparation was slowly stirred at roomtemperature for 30 minutes. POCl₃ (0.4 g, Aldrich, fresh-distilled, bp106-107° C.) was then added in two portions. The resulting preparationwas vigorously stirred with a glass rod for about 20 minutes. Theresulting gel was set at room temperature overnight, then thoroughlywashed with water to near neutral pH, and dried on a lyophilizer toprovide a white solid. Thus, crosslinked very high molecular weightdextran was produced.

AUL and CRC measurements of the product were then taken using a 0.9%NaCl solution as the liquid being absorbed. The crosslinked very highmolecular weight dextran was measured to have an AUL of 17.3 g/g under apsi (pounds-per-square inch) of 0.82, and a CRC of 23.1 g/g.

Crosslinking With Sodium Trimetaphosphate (STMP)

Very high molecular weight dextran (5 g) was dissolved in 50 mL water ina flask with a mechanical stirrer. Sodium hydroxide solution (10%, 1 g)was then added while stirring at about 159 rpm. An STMP solution (2.0 gof STMP in 10 mL DI water) was then added to the stirring preparation.After 1 hour of stirring, another 1.0 g of NaOH (10%) solution was addeddropwise. Again, after another hour elapsed, 1.0 g of NaOH (10%)solution was added dropwise. The stirring was adjusted to 51 rpm andcontinued overnight. The resulting material was washed with copiousamounts of water to a neutral pH, then dried under vacuum to give awhite solid. Thus, crosslinked very high molecular weight dextran wasproduced.

AUL and CRC measurements of the product were then taken using a 0.9%NaCl solution as the liquid being absorbed. The crosslinked very highmolecular weight dextran was measured to have an AUL of 13.3 g/g under apsi of 0.82, and a CRC of 12.2 g/g.

Crosslinking With Citric Acid

Very high molecular weight dextran (5.6 g) was added to water (48.5 g)portion by portion with stirring. The preparation was slowly stirreduntil a homogeneous viscous solution was formed. Citric acid (1.0 g) wasthen added, after which the preparation was dried in a vacuum oven at60° C. over 3 days. Water (5 mL) was added and the resulting preparationwas stirred at room temperature for 6 hours to provide a viscouspreparation. This preparation was lyophilized to provide a white solid.Thus, crosslinked very high molecular weight dextran was produced.

AUL and CRC measurements of the product were then taken using a 0.9%NaCl solution as the liquid being absorbed. The crosslinked very highmolecular weight dextran was measured to have an AUL of 7.1 g/g under apsi of 0.82, and a CRC of 9.5 g/g.

Crosslinking With Boric Acid

Boric acid (0.16 g) was dissolved in water (5 mL), after which NaOHsolution (2%, about 2.5 mL) was added to adjust the pH to about 10. Morewater was then added to bring the total solution volume to 14 mL. Veryhigh molecular weight dextran (0.97 g) was added to this solutionportion by portion with stirring. The preparation was allowed to set atroom temperature overnight, and then dried by lyophilization to providea solid. Thus, crosslinked very high molecular weight dextran wasproduced.

AUL and CRC measurements of the product were then taken using a 0.9%NaCl solution as the liquid being absorbed. The crosslinked very highmolecular weight dextran was measured to have an AUL of 14.2 g/g under apsi of 0.82, and a CRC of 17.2 g/g.

Crosslinking With Epichlorohydrin (ECH)

Very high molecular weight dextran (2.5 g) was added to water (17.5 g)portion by portion with stirring. The solution was slowly stirred untila homogeneous gel was formed. Sodium hydroxide solution (10%, 10 g) andwater 5 g were then added, after which ECH (0.84 g) was added. Thispreparation was stirred at room temperature. The resulting gel waswashed with water to near neutral pH. The washed gel was dried bylyophilization to provide a solid. Thus, crosslinked very high molecularweight dextran was produced.

AUL and CRC measurements of the product were then taken using a 0.9%NaCl solution as the liquid being absorbed. The crosslinked very highmolecular weight dextran was measured to have an AUL of 15.4 g/g under apsi of 0.82, and a CRC of 10.1 g/g.

Crosslinking With Divinyl Sulfone (DVS)

Very high molecular weight dextran (5.0 g) was added to 50 g of waterand 2.5 g of NaOH solution (2 wt%). This preparation was stirredovernight, after which DVS (0.225 g) in 4.5 g water was added withstirring. The viscosity of the preparation increased very rapidly afterthe addition of DVS. The preparation was allowed to set at roomtemperature over 3 days, after which it was washed with water to nearneutral pH, and then dried by lyophilization to provide a solid. Thus,crosslinked very high molecular weight dextran was produced.

AUL and CRC measurements of the product were then taken using a 0.9%NaCl solution as the liquid being absorbed. The crosslinked very highmolecular weight dextran was measured to have an AUL of 13 g/g under apsi of 0.82, and a CRC of 10.6 g/g.

Thus, very high molecular weight dextran was crosslinked using differentcrosslinking agents. Each of these crosslinked dextran materialsexhibited absorption of aqueous liquid, and are therefore contemplatedto be suitable for use in various aqueous liquid absorption applicationsas disclosed above.

1-15. (canceled)
 16. A composition comprising crosslinked dextran,wherein said dextran comprises: (i) about 87-91.5 wt% glucose linked atpositions 1 and 6; (ii) about 0.1-1.2 wt% glucose linked at positions 1and 3; (iii) about 0.1-0.7 wt% glucose linked at positions 1 and 4; (iv)about 7.7-8.6 wt% glucose linked at positions 1, 3 and 6; and (v) about0.4-1.7 wt% glucose linked at: (a) positions 1, 2 and 6, or (b)positions 1, 4 and 6; wherein one or more crosslinks of the crosslinkeddextran are covalent.
 17. The composition of claim 16, wherein saiddextran comprises: (i) about 89.5-90.5 wt% glucose linked only atpositions 1 and 6; (ii) about 0.4-0.9 wt% glucose linked only atpositions 1 and 3; (iii) about 0.3-0.5 wt% glucose linked only atpositions 1 and 4; (iv) about 8.0-8.3 wt% glucose linked only atpositions 1, 3 and 6; and (v) about 0.7-1.4 wt% glucose linked only at:(a) positions 1, 2 and 6, or (b) positions 1, 4 and
 6. 18. Thecomposition of claim 16, wherein the one or more crosslinks of thecrosslinked dextran comprise phosphorus.
 19. The composition of claim18, wherein the one or more crosslinks of the crosslinked dextrancomprise a phosphodiester bond.
 20. The composition of claim 16, whereinthe composition is a personal care product, household care product,medical product, or industrial product.
 21. The composition of claim 16,wherein the composition is a product selected from the group consistingof baby diapers, potty training pants, incontinence products, femininehygiene products, wound healing dressings, and sanitary towels.
 22. Thecomposition of claim 16, wherein the composition is a product selectedfrom the group consisting of (i) telecommunication cable wrapping, (ii)food pad, (iii) fire-fighting device, (iv) product for cleanup of acidicor basic aqueous solutions spills, and (v) agricultural or forestryproduct for retaining water in soil and/or to release water to plantroots.
 23. A composition comprising crosslinked dextran, wherein saiddextran is a product of a glucosyltransferase enzyme comprising an aminoacid sequence that is at least 90% identical to SEQ ID NO:1 or SEQ IDNO:2, and wherein one or more crosslinks of the crosslinked dextran arecovalent.
 24. The composition of claim 23, wherein said dextran is aproduct of a glucosyltransferase enzyme comprising an amino acidsequence that is at least 95% identical to SEQ ID NO:1 or SEQ ID NO:2.25. The composition of claim 23, wherein the one or more crosslinks ofthe crosslinked dextran comprise phosphorus.
 26. The composition ofclaim 25, wherein the one or more crosslinks of the crosslinked dextrancomprise a phosphodiester bond.
 27. The composition of claim 23, whereinthe composition is a personal care product, household care product,medical product, or industrial product.
 28. The composition of claim 23,wherein the composition is a product selected from the group consistingof baby diapers, potty training pants, incontinence products, femininehygiene products, wound healing dressings, and sanitary towels.
 29. Thecomposition of claim 23, wherein the composition is a product selectedfrom the group consisting of (i) telecommunication cable wrapping, (ii)food pad, (iii) fire-fighting device, (iv) product for cleanup of acidicor basic aqueous solutions spills, and (v) agricultural or forestryproduct for retaining water in soil and/or to release water to plantroots.